Alpha-amylase variant with altered properties

ABSTRACT

The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits altered properties relative to the parent alpha-amylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/086,558 filed Nov. 21, 2013 (now U.S. Pat. No. 9,080,137), which is acontinuation of U.S. application Ser. No. 13/455,905 filed Apr. 25, 2012(now U.S. Pat. No. 8,617,837), which is a continuation of U.S.application Ser. No. 13/020,545 filed Feb. 3, 2011 (now U.S. Pat. No.8,252,573), which is a continuation of U.S. application Ser. No.12/360,635 filed Jan. 27, 2009 (now U.S. Pat. No. 8,486,681), which is acontinuation of U.S. application Ser. No. 10/477,725 (now U.S. Pat. No.7,498,158) filed Nov. 14, 2003 which is a 35 U.S.C. 371 nationalapplication of PCT/DK02/00319 filed May 15, 2002, which claims priorityor the benefit under 35 U.S.C. 119 of Danish application nos. PA 200100760, PA 2001 00981, PA 2001 00982, PA 2001 00998, PA 2001 00999 and PA2001 01443 filed May 15, 2001, Jun. 22, 2001, Jun. 22, 2001, Jun. 26,2001, Jun. 26, 2001, and Oct. 2, 2001, respectively and of U.S.provisional application Nos. 60/296,631, 60/302,570, 60/302,391,60/302,392, 60/302,395, and 60/326,750, filed Jun. 7, 2001, Jul. 2,2001, Jun. 29, 2001, Jul. 2, 2001, Jul. 2, 2001, and Oct. 3, 2001,respectively, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a variant of parent Termamyl-likealpha-amylase, which variant has alpha-amylase activity and exhibits analteration in at least one of the following properties relative to saidparent alpha-amylase:

Substrate specificity, substrate binding, substrate cleavage pattern,thermal stability, pH activity profile, pH stability profile, stabilitytowards oxidation, Ca²⁺ dependency, reduced and increased pl andimproved wash performance, specific activity, stability under, e.g.,high temperature and/or low pH conditions, in particular at low calciumconcentrations. The variant of the invention are suitable for starchconversion, ethanol production, laundry wash, dish wash, hard surfacecleaning, textile desizing, and/or sweetner production.

BACKGROUND OF THE INVENTION

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a Termamyl-likealpha-amylase, which variant in comparison to the corresponding parentalpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylaseactivity and exhibits an alteration in at least one of the abovementioned properties relative to said parent alpha-amylase.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, alpha-amylase variants of the invention are described by useof the following nomenclature:

Original amino acid(s): position(s): substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

Ala30Asn or A30N

a deletion of alanine in the same position is shown as:

Ala30* or A30*

and insertion of an additional amino acid residue, such as lysine, isshown as:

Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific alpha-amylase contains a “deletion” in comparison withother alpha-amylases and an insertion is made in such a position this isindicated as:

*36Asp or *36D

for insertion of an aspartic acid in position 36.

Multiple mutations are separated by plus signs, i.e.:

Ala30Asp+Glu34Ser or A30N+E34S

representing mutations in positions 30 and 34 substituting alanine andglutamic acid for asparagine and serine, respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

A30N,E or

A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

Further, “A30X” means any one of the following substitutions:

A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M,A30F, A30P, A30S, A30T, A30W, A30Y, or A30V; or in short:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

If the parent enzyme—used for the numbering—already has the amino acidresidue in question suggested for substitution in that position thefollowing nomenclature is used:

-   -   “X30N” or “X30N,V” in the case where for instance one or N or V        is present in the wildtype.        Thus, it means that other corresponding parent enzymes are        substituted to an “Asn” or “Val” in position 30.        Characteristics of Amino Acid Residues        Charged Amino Acids:        Asp, Glu, Arg, Lys, His        Negatively Charged Amino Acids (with the Most Negative Residue        First):        Asp, Glu        Positively Charged Amino Acids (with the Most Positive Residue        First):        Arg, Lys, His        Neutral Amino Acids:        Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Met, Cys, Asn, Gln, Ser,        Thr, Pro        Hydrophobic Amino Acid Residues (with the Most Hydrophobic        Residue Listed Last):        Gly, Ala, Val, Pro, Met, Leu, Ile, Tyr, Phe, Trp,        Hydrophilic Amino Acids (with the Most Hydrophilic Residue        Listed Last):        Thr, Ser, Cys, Gln, Asn

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows an alignment of the amino acid sequences of five parentTermamyl-like alpha-amylases. The numbers on the extreme left designatethe respective amino acid sequences as follows:

1: SEQ ID NO: 4 (SP722)

2: SEQ ID NO: 2 (SP690)

3: SEQ ID NO: 10 (BAN)

4: SEQ ID NO: 8 (BLA)

5: SEQ ID NO: 6 (BSG).

DETAILED DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a Termamyl-likeamylase, which variant has alpha-amylase activity and exhibits analteration in at least one of the following properties relative to theparent alpha-amylase: substrate specificity, substrate binding,substrate cleavage pattern, thermal stability, pH activity profile, pHstability profile, stability towards oxidation, Ca²⁺ dependency,specific activity, stability under, e.g., high temperature and/or low pHconditions, in particular at low calcium concentrations.

Termamyl-Like Alpha-Amylases

A number of alpha-amylases produced by Bacillus spp. are highlyhomologous (identical) on the amino acid level.

The identity of a number of known Bacillus alpha-amylases can be foundin the below Table 1:

TABLE 1 Percent identity 707 AP1378 BAN BSG SP690 SP722 AA560 Termamyl707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.195.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.087.2 87.0 69.2 SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.586.0 66.9 66.3 87.0 86.8 100.0 68.3 Termamyl 68.1 69.4 80.7 65.4 69.270.8 68.3 100.0

For instance, the B. licheniformis alpha-amylase (BLA) comprising theamino acid sequence shown in SEQ ID NO: 8 (commercially available asTermamyl™) has been found to be about 81% homologous with the B.amyloliquefaciens alpha-amylase comprising the amino acid sequence shownin SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilusalpha-amylase (BSG) comprising the amino acid sequence shown in SEQ IDNO: 6. Further homologous alpha-amylases include SP690 and SP722disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ IDNO: 4, respectively, herein. Other amylases are the AA560 alpha-amylasederived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 anddescribed by Tsukamoto et al., Biochemical and Biophysical ResearchCommunications, 151 (1988), pp. 25-31.

The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (from KAOCorporation).

Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like alpha-amylases are comprised inthe products sold under the following tradenames: Optitherm™ andTakatherm™ (Solvay); Maxamyl™ (available from Gist-brocades/Genencor),Spezym AA™ and Spezyme Delta AA™ (available from Genencor), andKeistase™ (available from Daiwa), Dex lo, GC 521 (available fromGenencor) and Ultraphlow (from Enzyme Biosystems).

Because of the substantial homology found between these alpha-amylases,they are considered to belong to the same class of alpha-amylases,namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-like”alpha-amylase” is intended to indicate an alpha-amylase, in particularBacillus alpha-amylase, especially Bacillus licheniformis alpha-amylase,which, at the amino acid level, exhibits a substantial identity toTermamyl™, i.e., the B. licheniformis alpha-amylase having the aminoacid sequence shown in SEQ ID NO: 8, herein.

In other words, all the following alpha-amylases, which has the aminoacid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13 herein areconsidered to be “Termamyl-like alpha-amylase”. Other Termamyl-likealpha-amylases are alpha-amylases i) which displays at least 60%, suchas at least 70%, e.g., at least 75%, or at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% homology (identity) with at least one of said amino acidsequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, and/or isencoded by a DNA sequence which hybridizes to the DNA sequences encodingthe above-specified alpha-amylases which are apparent from SEQ ID NOS:1, 3, 5, 7, 9, and of the present specification (which encodingsequences encode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6,8, 10 and 12 herein, respectively).

Homology (Identity)

The homology may be determined as the degree of identity between the twosequences indicating a derivation of the first sequence from the second.The homology may suitably be determined by means of computer programsknown in the art such as GAP provided in the GCG program package(described above). Thus, Gap GCGv8 may be used with the default scoringmatrix for identity and the following default parameters: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3, respectively fornucleic acidic sequence comparison, and GAP creation penalty of 3.0 andGAP extension penalty of 0.1, respectively, for protein sequencecomparison. GAP uses the method of Needleman and Wunsch, (1970), J. Mol.Biol. 48, p. 443-453, to make alignments and to calculate the identity.

A structural alignment between Termamyl (SEQ ID NO: 8) and, e.g.,another alpha-amylase may be used to identify equiva-lent/correspondingpositions in other Termamyl-like alpha-amylases. One method of obtainingsaid structural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading(Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Hybridisation

The oligonucleotide probe used in the characterisation of theTermamyl-like alpha-amylase above may suitably be prepared on the basisof the full or partial nucleotide or amino acid sequence of thealpha-amylase in question.

Suitable conditions for testing hybridisation involve pre-soaking in5×SSC and prehybridizing for 1 hour at 40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation inthe same solution supplemented with 100 mM ATP for 18 hours at 40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular_Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate analpha-amylase, which is encoded by a DNA sequence of synthetic and/orcDNA origin and which has the identifying characteristics of thealpha-amylase in question. The term is also intended to indicate thatthe parent alpha-amylase may be a variant of a naturally occurringalpha-amylase, i.e., a variant, which is the result of a modification(insertion, substitution, deletion) of one or more amino acid residuesof the naturally occurring alpha-amylase.

Parent Termamyl-Like Alpha-Amylases

According to the invention any Termamy-like alpha-amylase, as definedabove, may be used as the parent (i.e., backbone) alpha-amylase. In apreferred embodiment of the invention the parent alpha-amylase isderived from B. licheniformis, e.g., one of those referred to above,such as the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8.

Parent Hybrid Termamyl-Like Alpha-Amylases

The parent alpha-amylase (i.e., backbone alpha-amylase) may also be ahybrid alpha-amylase, i.e., an alpha-amylase, which comprises acombination of partial amino acid sequences derived from at least twoalpha-amylases.

The parent hybrid alpha-amylase may be one, which on the basis of aminoacid homology (identity) and/or DNA hybridization (as defined above) canbe determined to belong to the Termamyl-like alpha-amylase family. Inthis case, the hybrid alpha-amylase is typically composed of at leastone part of a Termamyl-like alpha-amylase and part(s) of one or moreother alpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination ofpartial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives, may beany of the specific Termamyl-like alpha-amylase referred to herein.

For instance, the parent alpha-amylase may comprise a C-terminal part ofan alpha-amylase derived from a strain of B. licheniformis, and aN-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus (BSG). Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 10 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical tothe Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylaseshown in SEQ ID NO: 8, except that the N-terminal 35 amino acid residues(of the mature protein) has been replaced by the N-terminal 33 residuesof BAN (mature protein), i.e., the Bacillus amyloliquefaciensalpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segmentcorresponding to the 68 N-terminal amino acid residues of the B.stearothermophilus alpha-amylase having the amino acid sequence shown inSEQ ID NO: 6 and an amino acid segment corresponding to the 415C-terminal amino acid residues of the B. licheniformis alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8.

Another suitable parent hybrid alpha-amylase is the one previouslydescribed in WO 96/23874 (from Novo Nordisk) constituting the N-terminusof BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 ofthe mature protein) and the C-terminus from Termamyl (amino acids301-483 of the mature protein).

In a preferred embodiment of the invention the parent Termamyl-likealpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8 and SEQ ID NO:10. Specifically, the parent hybrid Termamyl-like alpha-amylase may be ahybrid alpha-amylase comprising the 445 C-terminal amino acid residuesof the B. licheniformis alpha-amylase shown in SEQ ID NO: 8 and the 37N-terminal amino acid residues of the alpha-amylase derived from B.amyloliquefaciens shown in SEQ ID NO: 10, which may suitably furtherhave the following mutations: H156Y+A181T+N190F+A209V+Q264S (using thenumbering in SEQ ID NO: 8). The latter mentioned hybrid is used in theexamples below and is referred to as LE174.

Other specifically contemplated parent alpha-amylase include LE174 withfewer mutations, i.e., the right above mentioned hydrid having thefollowing mutations: A181T+N190F+A209V+Q264S; N190F+A209V+Q264S;A209V+Q264S; Q264S; H156Y+N190F+A209V+Q264S; H156Y+A209V+Q264S;H156Y+Q264S; H156Y+A181T+A209V+Q264S; H156Y+A181T+Q264S; H156Y+Q264S;H156Y+A181T+N190F+Q264S; H156Y+A181T+N190F; H156Y+A181T+N190F+A209V.These hybrids are also considered to be part of the invention.

In a preferred embodiment the parent Termamyl-like alpha amylase isLE174, SP722, or AA560 including any of LE174+G48A+T49I+G107A+I201F;LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F, or SP722+D183*+G184*;SP722+D183*+G184*+N195F; SP722+D183*+G184*+M202L;SP722+D183*+G184*+N195F+M202L; BSG+I181*+G182*; BSG+I181*+G182*+N193F;BSG+I181*+G182*+M200L; BSG+I181*+G182*+N193F+M200L; AA560+D183*+G184*;AA560+D183*+G184*+N195F; AA560+D183*+G184*+M202L;AA560+D183*+G184*+N195F+M202L.

“BSG+I181*+G182*+N193F” means the B. stearothermophilus alpha-amylasehas been mutated as follows: deletions in positions I181 and G182 and asubstitution from Asn (N) to Phe (F) in position 193.

Other parent alpha-amylases contemplated include LE429, which is LE174with an additional substitution in I201F. According to the inventionLE335 is the alpha-amylase, which in comparison to LE429 has additionalsubstitutions in T49I+G107A; LE399 is LE335+G48A, i.e., LE174, withG48A+T49I+G107A+I201F.

Altered Properties

The following section describes the relationship between mutations,which are present in a variant of the invention, and desirablealterations in properties (relative to those of a parent Termamyl-likealpha-amylase), which may result therefrom.

As mentioned above the invention relates to a Termamyl-likealpha-amylase with altered properties.

Parent Termamyl-like alpha-amylaseS specifically contemplated inconnection with going through the specifically contemplated alteredproperties are the above mentioned parent Termamyl-like alpha-amylaseand parent hydrid Termamyl-like alpha-amylases.

The Bacillus licheniformis alpha-amylase (SEQ ID NO: 8) is used as thestarting point, but corresponding positions in, e.g., the SP722, BSG,BAN, AA560, SP690, KSM AP1378, #707 and other Termmayl-likealpha-amylases should be understood as disclosed and specificallycontemplated too.

In an aspect the invention relates to variant with altered properties asmentioned above.

In the first aspect a variant of a parent Termamyl-like alpha-amylase,comprising an alteration at one or more positions (using SEQ ID NO: 8for the amino acid numbering) selected from the group of:

5, 6, 36, 37, 38, 39, 42, 45, 47, 63, 66, 69, 70, 71, 72, 74, 75, 76,79, 82, 83, 86, 87, 89, 93, 112, 113, 117, 120, 137, 213, 216, 220, 223,225, 226, 227, 229, 243, 245, 279, 282, 311, 321, 324, 352, 353, 354,357, 361, 362, 364, 368, 390, 395, 397, 399, 400, 401, 425, 451, 452,453, 466, 468, 470, 471, 478,wherein(a) the alteration(s) are independently

(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position,

(ii) a deletion of the amino acid which occupies the position, or

(iii) a substitution of the amino acid which occupies the position witha different amino acid,

(b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 thepositions to be mutates are one or more of:

G5, T6, G36, I37, T38, A39, I42, A45, K47, D63, E66, Q69, K70, G71, T72,R74, T75, K76, T79, E82, L83, A86, I87, S89, R93, T112, E113, A117,V120, A137, K213, G216, A220, L223, L225, D226, G227, R229, D243, V245,F279, S282, T311, V321, V324, L352, T353, R354, G357, V361, F362, G364,G368, A390, A395, G397, Q399, H400, D401, A425, D451, I452, T453, G466,G468, F470, H471, S478.

In the first aspect the invention also relates to a variant of a parentTermamyl-like alpha-amylase, comprising a substitution at one or morepositions (using SEQ ID NO: 8 for the amino acid numbering) selectedfrom the group of:

7, 8, 9, 11, 12, 19, 21, 22, 25, 32, 40, 41, 46, 48, 55, 57, 58, 60, 77,95, 97, 98, 99, 100, 101, 102, 103, 105, 107, 115, 118, 135, 139, 141,143, 151, 159, 160, 161, 162, 163, 166, 175, 177, 183, 186, 187, 192,199, 200, 202, 203, 208, 212, 215, 219, 228, 230, 233, 236, 238, 240,241, 244, 248, 256, 258, 259, 260, 262, 270, 273, 274, 277, 281, 283,284, 285, 286, 287, 288, 289, 292, 295, 296, 304, 307, 312, 313, 320,322, 323, 325, 326, 478, 327, 329, 331, 339, 343, 344, 346, 347, 349,350, 359, 360, 369, 377, 380, 387, 409, 410, 411, 412, 423, 424, 426,427, 428, 429, 430, 438, 440, 441, 449, 462, 472, 477, 479, 480, 481wherein(a) the variant has alpha-amylase activity and (b) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 thepositions to be mutated are one or more of:

L7, M8, Q9, F11, E12, G19, H21, W22, L25, L32, V40, W41, Y46, G48, G55,G57, A58, D60, Y77, I95, V97, Y98, G99, D100, V101, V102, I103, H105,G107, V115, V118, I135, T139, F141, F143, S151, H159, F160, D161, G162,T163, D166, Y175, F177, D183, V186, S187, N192, A199, D200, D202, Y203,V208, I212, W215, Y219, F228, L230, V233, 1236, F238, F240, L241, W244,V248, M256, T258, V259, A260, Y262, L270, Y273, L274, T277, H281, V283,F284, D285, V286, P287, L288, H289, F292, A295, S296, M304, L307, V312,V313, S320, T322, F323, D325, N326, H327, T329, P331, V339, F343, K344,L346, A347, A349, F350, P359, Q360, T369, I377, L380, I387, V409, G410,W411, T412, G423, S478, L424, A426, L427, 1428, T429, D430, M438, V440,G441, W449, 1462, V472, V477, I479, Y480, V481

Specific Substitutions Contemplated are:

X7A,R,N,D,C,Q,E,G,H,K,M,P,S,Y,V;

X8C,M;

X9A,R,N,D,C,Q,G,H,M,P,S,T,W,Y,V;

X11A,N,D,C,Q,G,H,I,L,M,P,S,T,W,Y,V;

X12A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X19A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X21A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X22A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X25A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X32A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X40A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X41A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X46A,R,D,C,G,K,M,P,W,Y;

X48R,N,D,C,Q,E,G,H,K,M,F,P,W,Y;

X55A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X57R,N,D,C,Q,E,G,H,K,M,P,W;

X58A,R,N,D,C,Q,E,G,H,K,M,S,T,W,Y;

X60A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X77A,R,D,C,G,K,M,P,W,Y;

X95A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X97A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X98A,R,D,C,G,K,M,P,W,Y;

X99R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X100A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

X101A,N,C,Q,G,I,L,M,P,S,T,W,Y,V;

X102N,D,C,Q,E,H,I,L,M,F,P,W,Y,V;

X103A,N,D,C,Q,E,G,M,P,S,W,Y;

X105A,N,C,Q,G,H,I,L,M,P,S,T,Y,V;

X107R,N,D,Q,E,H,K,M,F,P,W,Y;

X115R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X118R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X135A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X139A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X141A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X143A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

X151A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

X159A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

X160A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X161A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X162A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X163A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X166A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X175A,R,D,C,G,K,M,P,W,Y;

X177A,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

X183A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X186A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X187A,R,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

X192A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X199R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X200A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X202A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X203A,R,D,C,G,K,M,P;

X208A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

X212A,N,C,Q,G,H,M,P,S,T,V;

X215A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X219A,R,D,C,G,K,M,P,W,Y;

X228A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

X230A,R,N,D,C,Q,E,G,L,M,P,S,T,W,Y,V;

X233R,N,C,Q,E,G,H,I,K,M,P,S,T,W,Y;

X236A,C,Q,G,H,I,M,P,S,T,V;

X238A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

X240A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X241A,N,C,Q,G,H,P,S,T,V;

X244A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X248A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X256C,M;

X258A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X259A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

X260R,N,D,C,Q,E,H,I,L,K,M,F,P,T,W,Y,V;

X262A,R,D,C,G,K,M,P,W,Y;

X270A,N,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

X273A,R,D,C,G,K,M,P,Y;

X274A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X277A,R,N,D,C,Q,E,G,H,K,M,P,S,W,Y,V;

X281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

X283A,R,N,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

X284A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,Y,V;

X285A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

X286A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V; preferably

X286N,C,Q,I,L,M,P,T,V,Y,F;

X287R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X288A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X289A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X292A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X295A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X296A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X304C,M;

X307A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X312A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X313A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X320R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X322R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

X323A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

X325A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X326A,R,N,C,Q,E,G,M,P,S,T,W;

X327A,R,C,G,H,I,L,K,M,P,S,T,W,Y,V;

X329A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X331N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X339A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X343A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

X344A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X346A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X347A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X349R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X350A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,Y,V;

X359R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X360N,D,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X369A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X377A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X380A,R,N,D,C,Q,E,G,H,K,P,S,W,Y,V;

X387A,R,N,D,C,Q,E,G,H,L,K,M,P,S,T,W,Y,V;

X409N,C,Q,E,G,H,M,P,S,T,W,Y,V;

X410A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X411A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X412R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X423A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X424A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X426A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X427A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

X428A,N,D,Q,E,G,H,I,M,F,P,S,W,Y,V;

X429A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X430A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X438C,M;

X440R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X441A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X449A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X462A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X472A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X477A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X479A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X480A,R,D,C,G,K,M,P,W,Y;

X481A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V

where each position corresponds to a position of the amino acid sequenceof the parent Termamyl-like alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

Also according to the first aspect, the invention relates to a variantof a parent Termamyl-like alpha-amylase, comprising a substitution atone or more positions (using SEQ ID NO: 8 for the amino acid numbering)selected from the group of:

1, 2, 3, 4, 13, 14, 16, 17, 18, 20, 23, 24, 26, 34, 35, 49, 50, 51, 52,53, 61, 62, 67, 68, 73, 84, 85, 88, 91, 92, 96, 106, 108, 114, 116, 119,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,136, 138, 145, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158,164, 165, 167, 168, 169, 170, 171, 172, 173, 176, 179, 180, 181, 182,184, 185, 188, 189, 190, 191, 193, 196, 198, 204, 205, 206, 209, 210,211, 214, 217, 218, 221, 222, 234, 235, 237, 239, 242, 246, 247, 249,250, 251, 252, 253, 254, 255, 257, 261, 263, 265, 266, 267, 268, 269,271, 272, 275, 276, 278, 280, 290, 291, 293, 294, 297, 298, 299, 300,301, 302, 303, 305, 306, 308, 309, 310, 314, 315, 316, 317, 318, 319,328, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, 345, 355, 358,363, 370, 371, 373, 374, 375, 376, 378, 379, 381, 389, 393, 394, 396,398, 402, 403, 404, 405, 406, 407, 408, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 431, 432, 433, 434, 435, 436, 437, 439, 442, 443,444, 445, 446, 447, 448, 450, 454, 455, 456, 457, 458, 459, 460, 461,463, 464, 465, 467, 469, 473, 474, 475, 476, 482, 483wherein(a) the variant has alpha-amylase activity and (b) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 thepositions to be mutated are one or more of:

A1, N2, L3, N4, W13, Y14, P16, N17, D18, Q20, R23, R24, Q26, E34, H35,T49, S50, Q51, A52, D53, L61, Y62, F67, H68, V73, Q84, S85, K88, H91,S92, N96, K106, G108, D114, T116, E119, D121, P122, A123, D124, R125,N126, R127, V128, I129, S130, G131, E132, H133, L134, K136, W138, G145,G147, S148, T149, Y150, D152, F153, K154, W155, H156, W157, Y158, D164,W165, E167, S168, R169, K170, L171, N172, R173, K176, G179, K180, A181,W182, W184, E185, N188, E189, N190, G191, Y193, L196, Y198, D204, H205,P206, A209, A210, E211, R214, T217, W218, N221, E222, K234, H235, K237,S239, R242, N246, H247, R249, E250, K251, T252, G253, K254, E255, F257,E261, W263, N265, D266, L267, G268, A269, E271, N272, N275, K276, N278,N280, Y290, Q291, H293, A294, T297, Q298, G299, G300, G301, Y302, D303,R305, K306, L308, N309, G310, S314, K315, H316, P317, L318, K319, D328,G332, Q333, S334, L335, E336, S337, T338, Q340, T341, W342, P345, E355,Y358, Y363, K370, G371, S373, Q374, R375, E376, P378, A379, K381, K389,Q393, Y394, Y396, A398, Y402, F403, D404, H405, H406, D407, 1408, R413,E414, G415, D416, S417, S418, V419, A420, N421, S422, G431, P432, G433,G434, A435, K436, R437, Y439, R442, Q443, N444, A445, G446, E447, T448,H450, G454, N455, R456, S457, E458, P459, V460, V461, N463, S464, E465,W467, E469, N473, G474, G475, S476, Q482, R483.

Specific Substitutions Contemplated are:

X1A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

X2R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X3A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

X4A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X13R,N,D,C,Q,E,G,H,K,M,P,S,T,W;

X14A,R,D,C,G,K,M,P,W;

X16R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X17A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X18A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X20A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X23A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

X24A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X26A,D,C,E,G,H,I,L,M,F,P,S,T,W,V;

X34A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X35A,R,N,D,C,Q,E,G,H,K,M,F,P,S,T,W,Y,V;

X49A,C,G,H,P,T;

X50A,R,N,C,Q,E,G,H,K,M,F,P,S,W;

X51A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X52A,R,D,C,Q,E,G,H,K,P;

X53A,D,C,G,H,K,M,P;

X61A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y;

X62A,R,D,C,G,K,M,P,Y;

X67A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

X68A,R,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X73A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

X84A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X85A,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X88A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X91A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X92A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X96A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X106A,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

X108R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X114A,N,C,Q,E,G,H,K,F,P,S,T,W,Y;

X116A,R,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

X119A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X121A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X122R,N,Q,G,H,I,L,M,F,S,T,W,Y,V;

X123N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X125R,N,Q,E,G,I,K,M,F,S,T,W,Y;

X126N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X127A,R,N,D,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X128A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,W,Y,V;

X129A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X130A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X131A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X132R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y;

X133R,N,D,C,M,T,W,V;

X134A,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X136A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X138R,N,D,Q,E,G,I,K,M,P,S,T,W,V;

X145A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

X147A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

X148A,R,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X149A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

X150A,D,C,G,M,P,W,Y;

X152A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X153A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X154A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X155A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

X157R,I,L,M,F,P,S,T,W,Y,V;

X158R,M,P,W,Y;

X164R,I,L,M,F,P,S,T,W,Y,V;

X165A,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X168A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,V;

X169A,R,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

X170A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X171A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

X172A,N,D,C,Q,E,G,I,L,M,F,P,T,W,Y,V;

X173A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

X176A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X179D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

X180A,G,I,L,M,F,P,W,Y,V;

X181G;

X182A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X184I,L,M,F,P,W,Y,V;

X185R,I,L,M,F,P,S,T,W,Y,V;

X188A,R,N,Q,G,H,L,M,F,W,V;

X189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

X190N;

X191A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X193A,R,G,M,P,W,Y;

X196A,N,Q,G,H,I,L,M,P,S,T,W,V;

X198A,R,G,M,P,W;

X204R,L,M,F,P,T,W,Y,V;

X205A,G,H,I,L,M,F,P,W,Y,V;

X206R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X209R,P,S,W,Y;

X210A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

X211E;

X214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

X217A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y;

X218A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X221A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X222A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X234A,D,C,G,H,I,K,M,F,P,S,T,W,Y,V;

X235A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X237A,G,H,I,L,M,F,W,Y,V;

X239G,H,I,L,M,F,P,S,T,Y,V;

X242G,I,L,M,F,S,T,W,Y,V;

X246A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X247R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

X249A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X250A,R,N,D,C,E,H,I,L,K,M,P,T,W,Y,V;

X251R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X252A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X253R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

X254A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

X257A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X261A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X263A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

X265C,Q,E,H,I,L,M,F,P,W;

X266A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X267A,R,N,D,C,Q,E,G,H,K,P,S,T,W,Y,V;

X268A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X269A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X271A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X272A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X275A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X276A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X278A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X280A,R,D,C,E,G,H,I,L,K,M,F,P,W,Y,V;

X290W,Y;

X291A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X293A,R,N,G,I,L,M,P,S,T,W,V;

X294R,N,Q,G,H,I,L,M,F,P,S,T,W,Y;

X297A,G,H,I,L,M,F,P,W,Y,V;

X298G,H,I,L,M,F,P,S,T,W,Y,V;

X299A,G,H,M,P,S,T;

X300A,C,G,H,I,L,M,F,P,T,W,Y,V;

X301N,Q,H,I,L,M,F,P,S,T,W,Y,V;

X302R,M,P,W,Y;

X303R,I,L,M,F,P,S,T,W,Y,V;

X305G,I,L,M,F,P,S,T,W,Y,V;

X306Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X308A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X309N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X310A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X314A,D,C,E,G,I,L,M,F,P,W,Y,V;

X315A,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X316A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

X317R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X318A,R,N,D,C,Q,E,G,H,I,K,P,S,W,Y,V;

X319A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X328A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X332A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X333A,N,D,C,G,I,M,F,P,S,T,Y,V;

X334R,N,C,Q,E,G,H,K,M,F,P,S,W,Y;

X335R,D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

X336A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X337A,R,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X338A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X340I,L,M,F,P,S,T,W,Y,V;

X341A,R,D,C,G,H,I,L,K,M,F,W,Y,V;

X342R,I,L,M,F,P,S,T,W,Y,V;

X345R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X358A,R,D,C,G,K,M,P,W,Y;

X363A,R,D,C,G,K,M,P,W,Y;

X370A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X371A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X373A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X374A,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X375A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,V;

X376A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X378R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X379A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

X381A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X389A,D,C,G,H,M,F,P,S,T,W,Y,V;

X393A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X394A,R,D,C,G,K,M,P,W,Y;

X396A,R,D,C,G,K,M,P,W,Y;

X398A,R,N,D,C,Q,E,G,H,I,L,M,F,W,Y,V;

X402R,C,G,K,M,P,W,Y;

X403A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X404R,I,L,M,F,P,S,T,W,Y,V;

X405R,G,H,I,L,M,F,P,W,Y,V;

X406A,R,G,H,I,M,F,P,Y,V;

X407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X408A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

X413A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X414A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X415A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X416A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X417A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

X418A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X419A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X420A,N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

X421A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X422A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X431A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X432G,H,I,L,M,F,P,S,T,W,Y,V;

X433A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X434A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X435Q,G,H,I,L,M,F,P,T,W,Y,V;

X436A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X437A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X439A,R,D,C,G,K,M,P,W,Y;

X442A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X443A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X444A,C,G,H,I,L,M,F,P,S,T,W,Y,V;

X445A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X446A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

X448A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

X454A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X455A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X456A,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

X457A,R,N,D,C,Q,E,G,H,I,L,K,M,F,W,Y,V;

X458A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X459R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y,V;

X460A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X461A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

X463A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

X464A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X465A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X467A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

X473N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X474A,R,G,H,I,L,M,F,P,W,Y,V;

X475A,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

X476G,H,I,L,M,F,P,S,T,W,Y,V;

X482A,N,D,C,G,H,I,L,M,F,S,T,W,Y,V;

X483A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V

where each position corresponds to a position of the amino acid sequenceof the parent Termamyl-like alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

Specifically contemplated according to the invention invention issubstitution at position 183 and/or 184 (using SEQ ID NO: 2 (SP690) forthe amino acid numbering) with any amino acid, i.e. any one of A, R, N,D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

Also in the first aspect the invention relates to a variant of a parentTermamyl-like alpha-amylase, comprising an alteration at one or morepositions (using SEQ ID NO: 8 for the amino acid numbering) selectedfrom the group of:

1, 3, 4, 17, 18, 20, 23, 24, 28, 56, 61, 62, 67, 68, 80, 81, 84, 85, 91,92, 106, 110, 114, 119, 121, 122, 123, 124, 125, 126, 127, 129, 131,134, 136, 172, 185, 196, 206, 217, 218, 231, 232, 235, 246, 247, 249,251, 257, 278, 310, 316, 317, 328, 332, 355, 358, 363, 367, 370, 373,375, 376, 381, 382, 391, 396, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 439, 445, 446, 448, 450, 454, 455, 458, 459, 460, 461, 463,464, 465, 467wherein(a) the alteration(s) are independently

(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position, or

(ii) a deletion of the amino acid which occupies the position,

(b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 thepositions to be mutated are one or more of:

A1, L3, N4, N17, D18, Q20, R23, R24, D28, Y56, L61, Y62, F67, H68, K80,G81, Q84, S85, H91, S92, K106, D110, D114, E119, D121, P122, A123, D124,R125, N126, R127, 1129, G131, L134, K136, N172, E185, L196, P206, T217,W218, D231, A232, H235, N246, H247, R249, K251, F257, N278, G310, H316,P317, D328, G332, E355, Y358, Y363, Y367, K370, S373, R375, E376, K381,H382, R391, Y396, R413, E414, G415, D416, S417, S418, V419, A420, N421,S422, Y439, A445, G446, T448, H450, G454, N455, E458, P459, V460, V461,N463, S464, E465, W467.

Specific insertions/deletions contemplated are:

A1 insertion;

L3 insertion;

N4 insertion;

N17 insertion;

D18 insertion;

Q20 insertion;

R23 insertion;

R24 insertion;

D28 insertion;

Y56 insertion;

L61 insertion or deletion;

Y62 insertion;

F67 insertion or deletion;

H68 insertion;

K80 insertion or deletion;

G81 insertion or deletion;

Q84 insertion;

S85 insertion;

H91 insertion or deletion;

S92 insertion or deletion;

K106 insertion or deletion;

D110 insertion or deletion;

D114 deletion;

E119 insertion or deletion;

D121 insertion;

P122 insertion;

A123 insertion;

D124 insertion;

R125 insertion;

N126 insertion;

R127 insertion;

I129 insertion;

G131 insertion;

L134 insertion;

K136 insertion;

N172 insertion;

E185 insertion;

L196 insertion or deletion;

P206 insertion or deletion;

T217 insertion;

W218 insertion;

D231 insertion or deletion;

A232 insertion or deletion;

H235 insertion or deletion;

N246 insertion;

H247 insertion;

R249 insertion;

K251 insertion;

F257 insertion or deletion;

N278 insertion;

G310 insertion or deletion;

H316 insertion;

P317 insertion;

D328 insertion or deletion;

G332 insertion or deletion;

E355 insertion or deletion;

Y358 insertion;

Y363 insertion;

Y367 insertion;

K370 insertion;

S373 insertion;

R375 insertion;

E376 insertion;

K381 insertion;

H382 insertion;

R391 insertion or deletion;

Y396 insertion;

R413 insertion or deletion;

E414 insertion or deletion;

G415 insertion or deletion;

D416 insertion;

S417 insertion;

S418 insertion;

V419 insertion;

A420 insertion;

N421 insertion;

S422 insertion or deletion;

Y439 insertion;

A445 insertion or deletion;

G446 insertion or deletion;

T448 insertion or deletion;

H450 insertion;

G454 insertion or deletion;

N455 insertion;

E458 insertion;

P459 insertion;

V460 insertion;

V461 insertion;

N463 insertion;

S464 insertion;

E465 insertion;

W467 insertion

where each position corresponds to a position of the amino acid sequenceof the parent Termamyl-like alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

Also in the first aspect the invention relates to a variant of a parentTermamyl-like alpha-amylase, comprising an alteration at one or morepositions (using SEQ ID NO: 8 for the amino acid numbering) selectedfrom the group of:

7, 8, 10, 11, 12, 15, 19, 21, 22, 25, 40, 41, 43, 44, 46, 55, 59, 77,78, 90, 95, 97, 98, 99, 100, 101, 102, 105, 109, 115, 118, 135, 139,141, 195, 208, 215, 219, 236, 238, 240, 244, 248, 256, 258, 259, 312,313, 320, 322, 323, 325, 326, 327, 330, 331, 348, 349, 350, 359, 360,365, 366, 369, 377, 384, 388, 423, 424, 438, 441, 449, 462, 479, 480,481wherein(a) the alteration(s) are independently

(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position, or

(ii) a deletion of the amino acid which occupies the position,

(b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 thepositions to be mutated are one or more of:

L7, M8, Y10, F11, E12, M15, G19, H21, W22, L25, V40, W41, P43, P44, Y46,G55, Y59, Y77, G78, L90, 195, V97, Y98, G99, D100, V101, V102, H105,A109, V115, V118, I135, T139, F141, Y195, V208, W215, Y219, I236, F238,F240, W244, V248, M256, T258, V259, V312, V313, S320, T322, F323, D325,N326, H327, Q330, P331, Y348, A349, F350, P359, Q360, D365, M366, T369,I377, I384, L388, G423, L424, M438, G441, W449, 1462, 1479, Y480, V481.

Specific insertions/deletions contemplated are:

L7 insertion or deletion;

M8 insertion;

Y10 insertion;

F11 insertion;

E12 insertion or deletion;

M15 insertion;

G19 insertion;

H21 insertion;

W22 insertion;

L25 insertion;

V40 insertion or deletion;

W41 insertion;

P43 insertion or deletion;

P44 insertion or deletion;

Y46 insertion;

G55 insertion;

Y59 insertion;

Y77 insertion;

G78 insertion or deletion;

L90 insertion or deletion;

I95 insertion;

V97 insertion;

Y98 insertion;

G99 insertion;

D100 insertion;

V101 insertion;

V102 insertion;

H105 insertion or deletion;

A109 insertion or deletion;

V115 insertion or deletion;

V118 insertion or deletion;

I135 insertion;

T139 insertion or deletion;

F141 insertion or deletion;

Y195 insertion;

V208 insertion or deletion;

W215 insertion;

Y219 insertion;

I236 insertion or deletion;

F238 insertion or deletion;

F240 insertion or deletion;

W244 insertion;

V248 insertion;

M256 insertion;

T258 insertion or deletion;

V259 insertion or deletion;

V312 insertion or deletion;

V313 insertion or deletion;

S320 insertion;

T322 insertion or deletion;

F323 insertion or deletion;

D325 insertion or deletion;

N326 insertion;

H327 insertion or deletion;

Q330 insertion or deletion;

P331 insertion or deletion;

Y348 insertion;

A349 insertion or deletion;

F350 insertion or deletion;

P359 insertion or deletion;

Q360 insertion;

D365 insertion or deletion;

M366 insertion;

T369 insertion;

I377 insertion;

I384 insertion or deletion;

L388 insertion or deletion;

G423 insertion or deletion;

L424 insertion or deletion;

M438 insertion;

G441 insertion or deletion;

W449 insertion;

I462 insertion;

I479 insertion or deletion;

Y480 insertion;

V481 insertion or deletion;

where each position corresponds to a position of the amino acid sequenceof the parent Termamyl-like alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

Corresponding positions in other parent alpha-amylases can be found byalignment as described above and shown in the alignment in FIG. 1.

Stability

In the context of the present invention, mutations (including amino acidsubstitutions and deletion) of importance with respect to achievingaltered stability, in particular improved stability (i.e., higher orlower), at especially high temperatures (i.e., 70-120° C.) and/orextreme pH (i.e. low or high pH, i.e, pH 4-6 or pH 8-11, respectively),in particular at free (i.e., unbound, therefore in solution) calciumconcentrations below 60 ppm, include any of the mutations listed in the“Altered Properties” section. The stability may be determined asdescribed in the “Materials & Methods” section below.

Ca²⁺ Stability

Altered Ca²⁺ stability means the stability of the enzyme under Ca²⁺depletion has been improved, i.e., higher or lower stability. In thecontext of the present invention, mutations (including amino acidsubstitutions and deletions) of importance with respect to achievingaltered Ca²⁺ stability, in particular improved Ca²⁺ stability, i.e.,higher or lower stability, at especially high pH (i.e., pH 8-10.5)include any of the mutations listed in the in “Altered properties”section.

Specific Activity

In a further aspect of the present invention, important mutations(including amino acid substitutions and deletions) with respect toobtaining variants exhibiting altered specific activity, in particularincreased or decreased specific activity, especially at temperaturesfrom 10-60° C., preferably 20-50° C., especially 30-40° C., include anyof the mutations listed in the in “Altered properties” section. Thespecific activity may be determined as described in the “Material &Methods” section below.

Oxidation Stability

Variants of the invention may have altered oxidation stability, inparticular higher oxidation stability, in comparison to the parentalpha-amylase. Increased oxidation stability is advantageous in, e.g.,detergent compositions and decreased oxidation stability may beadvantageous in composition for starch liquefaction. Oxidation stabilitymay be determined as described in the “Material & Methods” sectionbelow.

Altered pH Profile

Important positions and mutations with respect to obtaining variantswith altered pH profile, in particular improved activity at especiallyhigh pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) include mutations ofamino residues located close to the active site residues.

Preferred specific mutations/substitutions are the ones listed above inthe section “Altered Properties” for the positions in question. Suitableassays are described in the “Materials & Methods” section below.

Wash Performance

Important positions and mutations with respect to obtaining variantswith improved wash performance at especially high pH (i.e., pH 8.5-11)include the specific mutations/substitutions listed above in the section“Altered Properties” for the positions in question. The wash performancemay be tested as described below in the “Materials & Methods” section.

Increased pl

Substitutions Resulting in Higher pl

In an aspect the invention relates to a variant with a higher pl thanthe parent alpha-amylase. Such variants are suitable when adjusting thepl to the washing conditions of various detergents. This means that ifthe pl of the parent alpha-amylase is below the pH in the washingsolution the target is to increase the pl to the pH of the washingsolution. Such variant may be prepared by making the following kind ofsubstitutions:

1) Substituting one or more of the below mentioned negatively chargedamino acid residue in a parent alpha-amylase with a positively chargedamino acid residue.

2) Substituting one or more of the below mentioned neutral amino acidresidue in a parent alpha-amylase with a positively charged amino acidresidue;

3) Substituting one or more of the below mentioned negatively chargedamino acid residue in a parent alpha-amylase with a neutral amino acidresidue;

4) Substituting one or more of the below mentioned positively chargedamino acid residue in a parent alpha-amylase with a more positivelycharged amino acid residue;

Variants of the invention with increased pl in comparison to the parentalpha-amylase may have improved wash performance. Wash performance testsmay be carried out as described in the “Materials & Method” section.

Thus, variants of the invention include (using SEQ ID NO: 8 for thenumbering):

G5R,K,H;

T6R,K,H;

G36R,K,H;

I37R,K,H;

T38R,K,H;

A39R,K,H;

I42R,K,H;

A45R,K,H;

D63A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E66A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q69R,K,H;

G71R,K,H;

T72R,K,H;

T75R,K,H;

T79R,K,H;

E82A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L83R,K,H;

A86R,K,H;

I87R,K,H;

S89R,K,H;

T112R,K,H;

E113A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A117R,K,H;

V120R,K,H;

A137R,K,H;

G216R,K,H;

A220R,K,H;

L223R,K,H;

L225R,K,H;

D226A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G227R,K,H;

D243A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V245R,K,H;

F279R,K,H;

S282R,K,H;

T311R,K,H;

V321R,K,H;

V324R,K,H;

L352R,K,H;

T353R,K,H;

G357R,K,H;

V361R,K,H;

F362R,K,H;

G364R,K;H;

G368R,K,H;

A390R,K,H;

A395R, K, H;

G397R,K,H;

Q399R,K,H;

H400R,K,H;

D401A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A425R,K,H;

D451A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I452R,K,H;

T453R,K,H;

G466R,K,H;

G468R,K,H;

F470R,K,H;

S478R,K,H.

L7R,K,H;

Q9R,H;

F11H;

E12A,R,N,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G19R,K,H;

W22R,K,H;

L25R,K,H;

L32R,K,H;

V40R,K,H;

W41R,K,H;

Y46R,K;

G48R,K,H;

G55R,K,H;

G57R,K,H;

A58R,K,H;

D60A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y77R,K;

I95R,K,H;

V97R,K,H;

Y98R,K;

G99R,K,H;

D100A,R,C,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

V102H;

G107R,K,H;

V115R,K,H;

V118R,K,H;

I135R,K,H;

T139R,K,H;

F141R,K,H;

F143R,K,H;

S151R,K,H;

F160R,K,H;

D161R,K,H;

G162R,K,H;

T163R,K,H;

D166A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y175R,K;

F177K,H;

D183A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V186R,K,H;

S187R,K,H;

N192R,K,H;

A199R,K,H;

D200A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D202A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y203R,K;

V208R,K,H;

I212H;

W215R,K,H;

Y219R,K;

F228R,K,H;

L230R;

V233R,K,H;

I236H;

F238R,K,H;

F240R,K,H;

L241H;

W244R,K,H;

V248R,K,H;

T258R,K,H;

V259R,K,H;

A260R,K,H;

Y262R,K;

Y273R, K;

L274R,K,H;

T277R,K,H;

H281R,K;

V283R, K;

F284RK;

D285A,R,C,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

V286R,K,H;

P287R,H;

L288R,K,H;

H289R,K;

F292R,K,H;

A295R,K,H;

S296R,K,H;

L307R,K,H;

V312H;

V313R,K,H;

S320R,K,H;

T322R,K,H;

F323R,K;

D325A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N326R;

T329R,K,H;

P331H;

V339R,H,K;

F343R,H,K;

K344H;

L346R,K,H;

A347R,K,H;

A349R,K,H;

F350R,K,H;

P359R,K,H;

Q360H;

T369R,K,H;

I377R,K,H;

L380RK,H;

I387R,K,H;

V409H;

G410R,K,H;

W411R,K,H;

T412R,K,H;

G423R,K,H;

L424R,K,H;

A426R,K,H;

L427R,K,H;

I428H;

T429R,K,H;

D430A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V440R,K,H;

G441R,K,H;

W449R,K,H;

I462R,K,H;

V472R,K,H;

V477R,K,H;

I479R,K,H;

Y480R, K;

V481R,K,H.

A1R,K,H;

N2R,K,H;

L3R,K,H;

N4R,K,H;

W13R,K,H;

Y14R,K;

P16R,K,H;

N17R,K,H;

D18A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q20R,K,H;

R23H;

R24K,H;

Q26H;

E34A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

T49H;

S50R,K,H;

Q51H;

A52R,K,H;

D53A,C,G,H,K,M,P;

L61R,K,H;

Y62R,K;

F67R,K,H;

V73R,K,H;

Q84R,K,H;

S85R,K,H;

K88H;

S92R,H;

N96R,K,H;

K106H;

G108R,K,H;

D114A,N,C,Q,G,H,K,F,P,S,T,W,Y;

T116R,H;

E119A,R,N,D,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D121A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

P122R,H;

A123H;

D124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

R125K;

N126H;

R127K,H;

V128R,K,H;

I129R,K,H;

S130R,K,H;

G131R,K,H;

E132R,N,D,C,Q,G,H,I,L,K,M,F,S,W,Y;

L134K,H;

K136A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

W138R,K;

G145R,K,H;

G147R,K,H;

S148R,K,H;

T149R,K,H;

D152A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

F153R,K,H;

K154H;

W155R,K,H;

W157R;

Y158R;

D164R,I,L,M,F,P,S,T,W,Y,V;

W165K,H;

E167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S168R,K,H;

R169H;

K170H;

L171R,K,H;

R173H;

K176H;

G179K,H;

W182R,K,H;

E185R,I,L,M,F,P,S,T,W,Y,V;

N188R,H;

E189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

G191R,H;

Y193R;

L196H;

Y198R;

D204R,L,M,F,P,T,W,Y,V;

P206R,K,H;

A209R;

A210R,K,H;

T217R,H;

W218R,K,H;

N221R,K,H;

E222R,K,H;

K234H;

K237H;

S239H;

N246R,K,H;

R249H;

E250A,R,N,D,C,H,I,L,K,M,P,T,W,Y,V;

K251H;

T252R,K,H;

G253R,K,H;

K254H;

E255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

F257R,K,H;

E261A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

W263R,K;

N265H;

D266A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L267R,K,H;

G268R,K,H;

A269H;

E271A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N272R,K,H;

N275R,K,H;

K276H;

N278R,K,H;

N280R,K,H;

Q291R,H;

A294R,H;

T297H;

Q298H;

G299H;

G300H;

G301H;

Y302R;

D303R,I,L,M,F,P,S,T,W,Y,V;

K306H;

L308R,K,H;

N309H;

G310R,K,H;

K315H;

P317R,K,H;

L318R,K,H;

K319H;

D328A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G332R,K,H;

S334R,K,H;

L335R,K,H;

E336A,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

S337R,H;

T338R,K,H;

T341R,K,H;

W342R;

P345R,K,H;

E355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y358R,K;

Y363R,K;

K370H;

G371K,H;

S373R,K,H;

Q374K,H;

R375H;

E376A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

P378R,K,H;

A379R,K,H;

K381H;

K389H;

Q393K,H;

Y394R,K;

Y396R,M;

A398R,H;

Y402R,K;

F403R,H;

D404R,I,L,M,F,P,S,T,W,Y,V;

H406R;

D407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

I408R,K,H;

R413K,H;

E414A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G415R,K,H;

D416A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S417R,K,H;

S418R,K,H;

V419R,H;

A420K,H;

N421R,K,H;

S422R,K,H;

G431R,H;

P432H;

G433R,H;

G434R;

A435H;

K436H;

R437K,H;

Y439R, K;

R442H;

Q443R,H;

N444H;

A445R,K,H;

G446R,K,H;

E447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

T448R,K,H;

G454R,K,H;

N455R,K,H;

R456H;

S457R,K,H;

E458A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

P459R,K,H;

V460R,K,H;

V461R,H;

N463R,K,H;

S464R,K,H;

E465A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

W467R,K,H;

E469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N473H;

G474R,H;

G475H;

S476H;

Q482H;

R483H.

Decreased pl

Substitutions Resulting in Lower pl

Variants of the invention with decreased pl in comparison to the parentalpha-amylase may have improved liquefying effect. This means that thepl of the parent amylase should be adjusted to the pH conditions in theliquefying process in question. Normally the pH during liquefaction liesin the range of 4-7, such as between pH 4.5-6.5. An example of aliquefaction process is descripted below in the section “Liquefaction”.Improved liquefying effect may be carried out as described in the“Materials & Method” section.

Alternatively, variants with decreased pl may advantageously be used indetergent. If the pl of the parent alpha-amylase is above the pH in thewashing solution the target is to decrease the pl to the pH of thewashing solution. Such variant may be prepared by making the followingkind of substitutions:

1) Substituting one or more of the below mentioned positively chargedamino acid residue in a parent alpha-amylase with a neutral amino acidresidue.

2) Substituting one or more of the below mentioned neutral amino acidresidue in a parent alpha-amylase with a negatively charged amino acidresidue.

3) Substituting one or more of the below mentioned positively chargedamino acid residue with a negatively charged amino acid residue.

4) Substituting one or more of the below mentioned negatively chargedamino acid residue with a more negatively charged amino acid residue.

Thus, variants of the invention include (using SEQ ID NO: 8 for thenumbering):

G5D,E;

T6D,E;

G36D,E;

I37D,E;

T38D,E;

A39D,E;

I42D,E;

A45D,E;

K47A,R,N,D,C,Q,E,G,H,M,F,P,S,T,W,Y,V;

D63E;

Q69D,E;

K70A,N,D,C,Q,E,G,I,M,F,P,S,T,W,Y,V;

G71D,E;

T72D,E;

R74A,N,D,C,Q,E,G,I,M,F,P,S,T,W,Y,V;

T75D,E;

K76A,N,D,C,Q,E,G,L,M,F,P,S,T,W,Y,V;

T79D,E;

L83D,E;

A86D,E;

I87D,E;

S89D,E;

R93A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

T112D,E;

A117D,E;

V120D,E;

A137D,E;

K213A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

G216D,E;

A220D,E;

L223D,E;

L225D,E;

D226E;

G227D,E;

R229A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

D243E;

V245D,E;

F279D,E;

S282D,E;

T311D,E;

V321D,E;

V324D,E;

L352D,E;

T353D,E;

R354A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

G357D,E;

V361D,E;

F362D,E;

G364D,E;

G368D,E;

A390D,E;

A395D,E;

G397D,E;

Q399D,E;

H400A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

D401E;

A425D,E;

D451E;

I452D,E;

T453D,E;

G466D,E;

G468D,E;

F470D,E;

H471A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

S478D,E.

L7D,E;

Q9D;

F11D;

G19D,E;

H21R,D,E,K;

W22D,E;

L25D, E;

L32D,E;

V40D,E;

W41 D,E;

Y46D;

G48D,E;

G55D,E;

G57D,E;

A58D,E;

D60E;

Y77D;

I95D,E;

V97D,E;

Y98D;

G99D,E;

D100E;

V102D,E;

I103A,D,E;

H105A,N,C,Q,G,I,L,M,P,S,T,Y,V;

G107D,E;

V115D,E;

V118E;

I135D,E;

T139D,E;

F141D,E;

F143AD,E;

S151D,E;

H159A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

F160D,E;

D161E;

G162D,E;

T163D,E;

Y175D;

F177D,E;

D183E;

V186D,E;

S187E;

N192D,E;

A199D,E;

D200E;

D202E;

Y203D;

V208D,E;

W215D,E;

Y219D;

F228D,E;

L230D,E;

V233E;

F238D,E;

F240D,E;

W244D,E;

V248D,E;

T258D,E;

V259D,E;

A260D,E;

Y262D;

Y273D;

L274D,E;

T277D,E;

H281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

V283E;

F284D,E;

D285E;

V286D,E;

P287D,E;

L288D,E;

H289A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

F292D,E;

A295D, E;

S296D,E;

L307D,E;

V313D,E;

S320D,E;

T322D,E;

F323D,E;

D325E;

N326E;

H327A,R,C,G,I,L,K,M,P,S,T,W,Y,V;

T329D,E;

P331D,E;

V339E;

F343D,E;

K344A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

L346D,E;

A347D,E;

A349D,E;

P359D,E;

Q360D;

T369D,E;

I377D,E;

L380D,E;

I387D,E;

V409E;

G410D,E;

W411D,E;

T412E;

G423D,E;

L424D,E;

A426D,E;

L427D,E;

I428D,E;

T429D,E;

D430E;

V440D,E;

G441D,E;

W449D,E;

I462D,E;

V472D,E;

V477D,E;

I479D,E;

Y480D;

V481 D,E.

A1D,E;

N2D,E;

L3D,E;

N4D,E;

W13D,E;

Y14D;

P16D,E;

N17D,E;

D18E;

Q20D,E;

R23D,E;

R24D,E;

Q26D,E;

H35A,R,N,D,C,Q,E,G,K,M,F,P,S,T,W,Y,V;

S50E;

Q51D,E;

A52 D, E;

L61D,E;

Y62D;

F67D,E;

H68A,R,D,C,E,G,I,L,K,M,F,P,S,T,W,Y,V;

V73D,E;

Q84D;

S85E;

K88A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

H91A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

S92D,E;

N96D,E;

K106A,N,D,C,Q,E,G,I,L,M,P,S,T,Y,V;

G108D,E;

D114E;

T116D,E;

D121E;

A123D,E;

R125N,Q,E,G,I,M,F,S,T,W,Y;

R127D,E;

V128D;

I129D,E;

S130D,E;

G131D;

H133R,N,D,C,M,T,W,V;

L134D,E;

K136A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

W138D,E;

G145D,E;

G147D,E;

S148D,E;

T149D,E;

Y150D;

D152E;

F153D,E;

K154A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

W155D,E;

H156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

W165D,E;

S168D,E;

R169D,E;

K170A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

L171D,E;

N172D,E;

R173A,N,D,C,Q,E,G,M,P,S,W,Y,V;

K176A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

G179D,E;

K180A,G,I,L,M,F,P,W,Y,V;

W182D,E;

P206D,E;

A210D,E;

R214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

T217D;

W218D,E;

N221D,E;

K234A,D,C,G,I,M,F,P,S,T,W,Y,V;

H235A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

K237A,G,I,L,M,F,W,Y,V;

R242G,I,L,M,F,S,T,W,Y,V;

N246D,E;

H247R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

R249A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

K251R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

T252D,E;

G253E;

K254A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

F257D,E;

W263D,E;

N265E;

D266E;

L267D,E;

G268D,E;

N272D,E;

N275D,E;

K276A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

N278D,E;

N280D,E;

H293A,R,N,G,I,L,M,P,S,T,W,V;

R305G,I,L,M,F,P,S,T,W,Y,V;

K306Q,G,I,L,M,F,P,S,T,W,Y,V;

L308D,E;

G310E;

S314D,E;

K315A,N,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

H316A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

P317D,E;

L318D,E;

K319A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

D328E;

G332E;

Q333D;

S334E;

L335D,E;

S337E;

T341D;

P345D, E;

Y358D;

Y363D;

K370A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

G371E;

S373D,E;

Q374D,E;

R375A,N,D,C,Q,G,I,L,M,F,P,S,T,W,V;

P378D,E;

A379D,E;

K381A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

K389A,D,C,G,M,F,P,S,T,W,Y,V;

Q393E;

Y394D;

Y396D;

A398D,E;

H405R,G,I,L,M,F,P,W,Y,V;

H406A,R,G,I,M,F,P,Y,V;

I408D,E;

R413A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

G415D,E;

D416E;

S417D,E;

S418D,E;

V419D,E;

A420D,E;

N421D,E;

S422D,E;

K436A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

R437A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

Y439D;

R442A,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

Q443D,E;

A445D,E;

G446D,E;

T448D,E;

H450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

G454D,E;

R456A,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

S457D,E;

P459D,E;

V460D,E;

V461D,E;

N463D,E;

S464D,E;

W467D,E;

Q482D;

R483A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V.

Reduced Sensitivity to Anionic Surfactants

Substitutions Resulting in a More Hydrophilic Amino Acid Residue

In an aspect, the invention relates to providing alpha-amylase variantswith reduced sensitivity (or improved stability against denaturation) toanionic surfactants (in particular linear alkyl sulphonates (LAS)).These variants are provided by substituting, deleting or inserting anamino acid residue in the parent alpha-amylase as indicated below with amore hydrophilic amino acid residue. Such variants may be prepared by:

1) Substituting one or more of the below mentioned positively chargedamino acid residue in a parent alpha-amylase with a hydrophilic aminoacid residue.

2) Substituting one or more of the below mentioned hydrophobic aminoacid residue in a parent alpha-amylase with a hydrophilic amino acidresidue

3) Substituting one or more of the below mentioned positively chargedamino acid residue in a parent alpha-amylase with a netral or negativelycharged amino acid residue.

The anionic surfactants (in particular linear alkyl sulphonates (LAS))sensitivity (in detergent) may be tested as described in the “Materials& Methods” section.

Variants of the invention with reduced sensitivity to anionicsurfactants, in particular linear alkyl sulphonates (LAS), include(using the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8numbering):

G5N,C,Q,S,T;

T6N,C,Q,S;

G36N,C,Q,S,T;

I37N,C,Q,S,T;

T38N,C,Q,S;

A39N,C,Q,S,T;

I42N,C,Q,S,T;

A45N,C,Q,S,T;

K47N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

Q69N;

K70N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

G71N,D,C,Q,S,T;

T72N,C,Q,S;

R74N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

T75N,C,Q,S;

K76N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

T79N,C,Q,S;

L83N,C,Q,S,T;

A86N,C,Q,S,T;

I87N,C,Q,S,T;

S89N,C,Q;

R93N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

T112N,C,Q,S;

A117N,C,Q,S,T;

V120N,C,Q,S,T;

A137N,C,Q,S,T;

K213N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

G216N,C,Q,S,T;

A220N,C,Q,S,T;

L223N,C,Q,S,T;

L225N,C,Q,S,T;

G227N,C,Q,S,T;

R229N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

V245N,C,Q,S,T;

F279N,C,Q,S,T;

S282N,C,Q;

T311N,C,Q,S;

V321N,C,Q,S,T;

V324N,C,Q,S,T;

L352N,C,Q,S,T;

T353N,C,Q,S;

R354N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

G357N,C,Q,S,T;

V361N,C,Q,S,T;

F362N,C,Q,S,T;

G364N,C,Q,S,T;

G368N,C,Q,S,T;

A390N,C,Q,S,T;

A395N,C,Q,S,T;

G397N,C,Q,S,T;

Q399N;

H400N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

A425N,C,Q,S,T;

I452N,C,Q,S,T;

T453N,C,Q,S;

G466N,C,Q,S,T;

G468N,C,Q,S,T;

F470N,C,Q,S,T;

H471N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

S478N,C,Q.

L7N,C,Q,S;

M8C;

Q9N;

F11N,C,Q,S,T;

G19N,C,Q,S,T;

H21N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

W22N,C,Q,S,T;

L25N,C,Q,S,T;

L32N,C,Q,S,T;

V40N,C,Q,S,T;

W41N,C,Q,S,T;

Y46C;

G48N,C,Q;

G55N,C,Q,S,T;

G57N,C,Q;

A58N,C,Q,S,T;

Y77C;

I95N,C,Q,S,T;

V97N,C,Q,S,T;

Y98C;

G99N,C,Q,S,T;

V101N,C,Q,S,T;

V102N,C,Q;

I103N,C,Q,S;

H105N,C,Q,S,T,A,I,L,M,P,Y,V;

G107N,Q;

V115N,C,Q,S,T;

V118N,C,Q,S,T;

I135N,C,Q,S,T;

T139N,C,Q,S;

F141N,C,Q,S,T;

F143N,C,Q,S,T;

S151N,C,Q;

H159N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

F160N,C,Q,S,T;

G162N,C,Q,S,T;

T163N,C,Q,S;

Y175C;

F177N,C,Q,S,T;

V186N,C,Q,S,T;

S187C,Q;

A199N,C,Q,S,T;

Y203C;

V208N,C,Q,S,T;

I212N,C,Q,S,T;

W215N,C,Q,S,T;

Y219C;

F228N,C,Q,S,T;

L230N,C,Q,S,T;

V233N,C,Q,S,T;

I236C,Q,S,T;

F238N,C,Q,S,T;

F240N,C,Q,S,T;

L241N,C,Q,S,T;

W244N,C,Q,S,T;

V248N,C,Q,S,T;

M256C;

T258N,C,Q,S;

V259N,C,Q,S,T;

A260N,C,Q,T;

Y262C;

L270N,C,Q,S,T;

Y273C;

L274N,C,Q,S,T;

T277N,C,Q,S;

H281N,C,Q,S,T,A,M,P,W,Y,V,D,E;

V283N,C,Q,S,T;

F284N,C,Q,S,T;

V286C,Q,S,T;

P287N,C,Q,S,T;

L288N,C,Q,S,T;

H289N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

F292N,C,Q,S,T;

A295N,C,Q,S,T;

S296N,C,Q,T;

M304C;

L307N,C,Q,S,T;

V312N,C,Q,S,T;

V313N,C,Q,S,T;

S320N,C,Q;

T322N,C,Q,S;

F323N,C,Q,S,T;

H327C,S,T,A,I,L,M,P,W,Y,V;

T329N,C,Q;

P331N,C,Q,S,T;

V339N,C,Q,S,T;

F343N,C,Q,S,T;

K344N,C,Q,S,T;

L346N,Q,S,T;

A347N,C,Q,S,T;

A349N,Q,S,T;

F350N,C,Q,S,T;

P359N,C,Q,S,T;

Q360N;

T369N,C,Q,S;

I377N,C,Q,S,T;

L380N,C,Q,S;

I387N,C,Q,S,T;

V409N,C,Q,S,T;

G410N,C,Q,S,T;

W411N,C,Q,S,T;

T412N,C,Q,S;

G423N,C,Q,S,T;

L424N,C,Q,S,T;

A426N,C,Q,S,T;

L427N,C,Q,S,T;

I428N,Q,S;

T429N,C,Q,S;

M438C;

V440N,C,Q,S,T;

G441N,C,Q,S,T;

W449N,C,Q,S,T;

I462N,C,Q,S,T;

V472N,C,Q,S,T;

V477N,C,Q,S,T;

I479N,Q,S;

Y480C;

V481N,C,Q,S,T.

A1N,C,Q,S,T;

L3N,C,Q,S,T;

N4C,Q,S,T;

W13N,C,Q,S,T;

Y14C;

P16N,C,Q,S,T;

Q20N;

R23N,C,Q,S,A,I,L,M,F,P,W,Y,V,D,E;

R24N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

H35N,C,Q,S,T,A,M,F,P,W,Y,V,D,E;

T49C;

S50N,C,Q;

Q51N;

A52C,Q;

L61N,C,Q,S,T;

Y62C;

F67N,C,Q,S,T;

H68C,S,T,A,I,L,M,F,P,W,Y,V,D,E;

V73N,C,Q,S,T;

Q84N;

S85N,C,T;

K88N,C,S,T,A,I,L,M,F,P,W,Y,V,D,E;

H91N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

S92N,C,Q;

K106N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

G108N,C,Q,S,T;

T116C,Q,S;

E119N,Q,S,T;

P122N,Q,S,T;

A123N,C,Q,S,T;

R125N,Q,S,T,I,M,F,W,Y,E;

R127N,S,T,A,I,L,M,F,P,W,Y,V,D,E;

V128N,C,Q,S;

I129N,C,Q,S;

S130N,Q;

G131N,C,Q,S,T;

H133N,C,T,M,W,V,D;

L134N,C,S,T;

K136N,C,S,T A,I,L,M,F,P,W,Y,V,D,E;

W138N,Q,S,T;

G145N,C,Q,S,T;

G147N,C,Q,S,T;

S148C,Q,T;

T149N,C,Q,S;

Y150C;

F153N,C,Q,S,T;

K154N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

W155N,C,Q,S,T;

H156C,Q,S,T A,I,L,M,F,P,W,V,E;

W157S,T;

W165N,C,Q,S,T;

S168N,C,Q;

R169AN,C,Q,S A,M,P,W,Y,V,D,E;

K170N,C,S,T A,I,L,M,F,P,W,Y,V,D,E;

L171N,C,Q,S,T;

R173N,C,Q,S A,M,P,W,Y,V,D,E;

K176N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

G179C,Q;

W182N,C,Q,S,T;

K180A,I,L,M,F,P,W,Y,V;

W182N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

W184I,L,M,F,P,W,Y,V;

G191N,Q,S,T;

L196N,Q,S,T;

P206N,C,Q,S,T;

A209S;

A210N,C,Q;

R214C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

T217N,C,Q,S;

W218N,C,Q,S,T;

K234C,S,T A,I,M,F,P,W,Y,V,D;

H235N,C,Q,S,T A,I,L,M,F,P,W,Y,V;

R242S,T,I,L,M,F,W,Y,V;

H247N,C,Q,S,T,I,L,M,F,P,W,V;

R249N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

K251N,C,S,T I,L,M,F,P,W,Y,V,D,E;

T252N,C,Q,S;

G253C,Q,S,T;

K254N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

F257N,C,Q,S,T;

W263N,C,Q,S,T;

L267N,C,Q,S,T;

G268N,C,Q,S,T;

A269N,C,Q,S,T;

K276N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

Q291N;

H293N,S,T A,I,L,M,P,W,V;

A294N,Q,S,T;

G299S,T;

G300C,T;

G301N,Q,S,T;

R305S,T,I,L,M,F,P,W,Y,V;

K306Q,S,T,I,L,M,F,P,W,Y,V;

L308N,C,Q,S,T;

G310N,C,Q,S,T;

S314C;

K315N,C,Q,S,T A,I,L,M,F,P,W,Y,V,E;

H316N,C,Q,S,T A,I,L,M,F,P,W,V,D,E;

P317N,C,Q,S,T;

L318N,C,Q,S;

K319N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

G332N,C,Q,S,T;

Q333N;

S334N,C,Q;

L335C,Q;

S337N,C,Q;

T338N,C,Q,S;

T341C;

W342S,T;

P345N,C,Q,T;

E355N,C,Q,S,T;

Y358C;

Y363C;

K370N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

G371N,C,Q,S,T;

S373N,C,Q,T;

Q374N;

R375N,C,Q,S,T A,I,L,M,F,P,W,V,D;

P378N,C,Q,S,T;

A379N,C,Q,T;

K381N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

K389C,S,T,A,M,F,P,W,Y,V,D;

Q393N;

Y394C;

Y396C;

A398N,C,Q;

Y402C;

F403N,Q,S,T;

H405I,L,M,F,P,W,Y,V;

H406A,I,M,F,P,Y,V;

I408N,C,Q,S,T;

R413N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

G415N,C,Q,S,T;

S417N,Q;

S418N,Q;

V419N,C,Q,S,T;

A420N,C,S,T;

S422N,Q;

G431N,Q,S,T;

P432S,T;

G433N,Q,S,T;

G434N,Q,S,T;

A435Q,T;

K436N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

R437N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

Y439C;

R442N,C,S,T,A,I,L,M,F,P,W,Y,V,D,E;

Q443N;

A445N,C,Q,S,T;

G446N,C,Q,S,T;

T448N,C,Q,S;

H450N,C,Q,S,T,A,I,L,M,F,P,W,V,D,E;

G454N,C,Q,S,T;

R456C,S,T,A,I,L,M,F,P,W,Y,V,D,E;

S457N,C,Q;

P459N,C,Q,S;

V460N,C,Q,S,T;

V461N,C,Q,S;

S464N,C,Q,T;

W467N,C,Q,S,T;

G475N,Q,S,T;

Q482N;

R483N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E.

Increased Stability at Low pH and/or at High Temperature

Substitutions Resulting in a More Hydrophobic Amino Acid Residue

In an aspect the invention relates to Termamyl-like alpha-amylasevariant with increased stability at acidic pH and/or at high temperaturein comparison to the parent alpha-amylase. Such variants are especiallysuitable for starch liquefaction.

In the context of the invention the term “acidic pH” means a pH below7.0, especially below the pH range, in which industrial starchliquefaction processes are normally performed, which is between pH 5.5and 6.2.

In the context of the invention “high temperature” is a temperature inthe range from 60-110° C.

Such variant are variant having a substitution resulting in a morehydrophobic amino acid residues. Providing such variant of the inventionmay be prepared by

1) Substituting a charged amino acid residue with a hydrophobic aminoacid residue;

2) Substituting a hydrophilic amino acid residue with a hydrophobicamino acid residue;

3) Substituting a hydrophilic amino acid residue with a more hydrophilicamino acid residue;

4) Substituting a hydrophilic amino acid residue with a less hydrophilicamino acid residue.

Thus, variants of the invention include (using SEQ ID NO: 8 for thenumbering):

G5A,V,P,M,L,I,Y,F,W;

T6G,A,V,P,M,L,I,Y,F,W;

G36A,V,P,M,L,I,Y,F,W;

I37Y,F,W;

T38G,A,V,P,M,L,I,Y,F,W;

A39V,P,M,L,I,Y,F,W;

I42Y,F,W;

A45V,P,M,L,I,Y,F,W;

K47G,A,I,L,M,F,P,W,Y,V;

D63 G,A,V,P,M,L,I,Y,F,W;

E66G,A,V,P,M,L,I,Y,F,W;

Q69T,S,C, G,A,V,P,M,L,I,Y,F,W;

K70G,A,V,P,M,L,I,Y,F,W;

G71A,V,P,M,L,I,Y,F,W;

T72G,A,V,P,M,L,I,Y,F,W;

R74G,A,V,P,M,L,I,Y,F,W;

T75G,A,V,P,M,L,I,Y,F,W;

K76G,A,V,P,M,L,I,Y,F,W;

T79G,A,V,P,M,L,I,Y,F,W;

E82 G,A,V,P,M,L,I,Y,F,W;

L83I,Y,F,W;

A86V,P,M,L,I,Y,F,W;

I87Y,F,W;

S89G,A,V,P,M,L,I,Y,F,W;

R93 G,A,V,P,M,L,I,Y,F,W;

T112 G,A,V,P,M,L,I,Y,F,W;

E113 G,A,V,P,M,L,I,Y,F,W;

A117,V,P,M,L,I,Y,F,W;

V120P,M,L,I,Y,F,W;

A137V,P,M,L,I,Y,F,W;

K213G,A,V,P,M,L,I,Y,F,W;

G216A,V,P,M,L,I,Y,F,W;

A220V,P,M,L,I,Y,F,W;

L2231,Y,F,W;

L2251,Y,F,W;

D226G,A,V,P,M,L,I,Y,F,W;

G227G,A,V,P,M,L,I,Y,F,W;

R229G,A,V,P,M,L,I,Y,F,W;

D243G,A,V,P,M,L,I,Y,F,W;

V245P,M,L,I,Y,F,W;

F279W;

S282T,G,A,V,P,M,L,I,Y,F,W;

T311 G,A,V,P,M,L,I,Y,F,W;

V321P,M,L,I,Y,F,W;

V324P,M,L,I,Y,F,W;

L3521,Y,F,W;

T353G,A,V,P,M,L,I,Y,F,W;

R354G,A,V,P,M,L,I,Y,F,W;

G357A,V,P,M,L,I,Y,F,W;

V361P,M,L,I,Y,F,W;

F362W;

G364A,V,P,M,L,I,Y,F,W;

G368A,V,P,M,L,I,Y,F,W;

A390V,P,M,L,I,Y,F,W;

A395V,P,M,L,I,Y,F,W;

G397A,V,P,M,L,I,Y,F,W;

Q399T,S,C,G,A,V,P,M,L,I,Y,F,W;

H400G,A,V,P,M,L,I,Y,F,W;

D401 G,A,V,P,M,L,I,Y,F,W;

A425V,P,M,L,I,Y,F,W;

D451G,A,V,P,M,L,I,Y,F,W;

I452Y,F,W;

T453G,A,V,P,M,L,I,Y,F,W;

G466G,A,V,P,M,L,I,Y,F,W;

G468A,V,P,M,L,I,Y,F,W;

F470W;

H471 G,A,V,P,M,L,I,Y,F,W;

S478T,G,A,V,P,M,L,I,Y,F,W

Q9A,C,G,M,P,S,T,W,Y,V;

F11W;

E12A,G,I,L,M,F,P,W,Y,V;

G19A,I,L,M,F,P,W,Y,V;

H21A,G,I,L,M,F,P,W,Y,V;

L25I,W,Y;

L32I,F,W,Y;

V40I,L,M,F,P,W,Y;

Y46W;

G48M,F,P,W,Y;

G55AI,L,M,F,P,W,Y,V;

G57M,P,W;

A58G,M,W,Y;

D60AG,I,L,M,F,PW,Y,V;

Y77W;

I95F,W,Y;

V97I,L,M,F,P,W,Y;

Y98W;

G99I,L,M,F,P,W,Y,V;

D100A,G,I,M,F,P,W,Y,V;

V101I,L,M,P,W,Y;

V102I,L,M,F,P,W,Y;

I103W;

H105A,G,I,L,M,P,Y,V;

G107M,F,P,W,Y;

V115I,L,M,F,P,W,Y;

V118I,L,M,F,P,W,Y;

I135F,W,Y;

T139AG,I,L,M,F,P,W,Y,V;

F141W;

S151A,G,M,P,T,Y,V;

H159A,G,I,L,M,F,P,W,V;

F160W;

D161A,G,I,L,M,F,P,W,Y,V;

G162A,I,L,M,F,P,W,Y,V;

T163A,G,I,L,M,F,P,Y,V;

D166A,G,I,L,M,F,P,W,Y,V;

Y175W;

F177W;

D183A,G,I,L,M,F,P,W,Y,V;

V186I,L,M,F,P,W,Y;

S187A,G,I,L,M,F,P,W,Y,V;

N192A,G,I,L,M,F,P,W,Y,V;

A199G,I,L,M,F,P,W,Y,V;

D200AG,I,L,M,F,P,W,Y,V;

D202A,G,I,L,M,F,P,W,Y,V;

V208L,M,F,P,W,Y;

Y219W;

F228W;

L230W,Y;

V233G,I,M,P,W,Y;

I236M,P;

F238W;

F240W;

L241P;

V248A,G,I,L,M,F,P,W,Y;

T258A,G,I,M,F,P,W,Y,V;

V259M,P,W,Y;

A260I,L,M,F,P,W,Y,V;

Y262W;

L270I,F,W,Y;

L274I,M,F,P,W,Y;

T277A,G,M,P,W,Y,V;

H281A,G,M,P,W,Y,V;

V283I,L,M,F,P,W,Y;

D285A,G,I,M,F,P,W,Y,V;

V286I,M,F,P,W,Y;

P287I,L,M,F,W,Y;

L288I,F,W,Y;

H289A,G,I,L,M,F,P,W,Y,V;

F292W;

A295I,L,M,F,P,W,Y,V;

S296A,G,I,L,M,F,P,W,T,Y,V;

L307I,F,W,Y;

V312I,L,M,F,P,W,Y;

V313I,L,M,F,P,W,Y;

S320G,I,L,M,F,P,T,W,Y,V;

T322G,L,M,F,P,W,Y,V;

F323W;

D325AG,I,L,M,F,P,W,Y,V;

N326A,C,G,M,P,S,T,W;

H327A,G,I,L,M,P,W,Y,V;

T329A,G,I,L,M,F,P,W,Y,V;

P331I,L,M,F,W,Y;

V339I,L,M,F,P,W,Y;

K344AG,I,L,M,F,P,W,Y,V;

L346I,F,W,Y;

A347I,L,M,F,P,W,Y,V;

A349I,L,M,F,P,W,Y,V;

P359I,L,M,F,W,Y;

Q360I,L,M,F,P,S,T,W,Y,V;

T369A,G,I,L,M,F,P,W,Y,V;

I377F,W,Y;

L380W,Y;

I387W,Y;

V409M,P,W,Y;

G410A,I,L,M,F,P,W,Y,V;

T412G,I,L,M,F,P,W,Y,V;

G423A,I,L,M,F,P,W,Y,V;

L424I,F,W,Y;

A426I,L,M,F,P,W,Y,V;

L427Y;

I428F,W,Y;

T429A,G,I,L,M,F,P,W,Y,V;

D430A,G,I,L,M,F,P,W,Y,V;

V440I,L,M,F,P,W,Y;

G441A,I,L,M,F,P,W,Y,V;

I462F,W,Y;

V472I,L,M,F,P,W,Y;

V477I,L,M,F,P,W,Y;

I479F,W,Y;

Y480W;

V481M,P,Y.

A1I,L,M,F,P,W,Y;

N2C,Q,I,L,M,F,S,T,W,Y,V;

L3I,M,F,P,W,Y;

N4A,C,Q,I,L,M,F,P,S,T,W,Y,V;

Y14W;

P16I,L,M,F,W,Y;

N17A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

D18A,G,I,L,M,F,P,W,Y,V;

Q20A,C,I,L,M,F,P,S,T,W,Y,V;

R23A,G,I,L,M,F,P,W,Y,V;

R24A,G,I,L,M,F,P,W,Y,V;

Q26A,C,G,I,L,M,F,P,S,T,W,V;

E34A,G,I,L,M,F,P,W,Y,V;

H35A,G,M,F,P,W,Y,V;

T49A,G,P;

S50A,G,M,F,P,W;

Q51A,C,G,I,L,M,F,P,S,T,W,Y,V;

A52P;

D53A,G,M,P;

L61I,M,Y;

H68A,G,I,L,M,F,P,W,Y,V;

V73M,P,W,Y;

Q84A,C,G,I,L,M,F,P,S,T,W,Y,V;

S85A,G,I,L,M,F,P,T,W,Y,V;

K88A,G,I,L,M,F,P,W,Y,V;

H91A,G,I,L,M,F,P,W,Y,V;

S92A,G,I,L,M,F,P,T,W,Y,V;

N96A,C,G,I,L,M,F,P,S,T,W,Y,V;

K106A,G,I,L,M,P,Y,V;

G108I,L,M,F,P,W,Y,V;

D114A,G,F,P,W,Y;

T116A,G,I,L,M,F,P,W,Y,V;

E119A,G,I,L,M,F,P,W,Y,V;

D121A,G,I,L,M,F,P,W,Y,V;

P122I,L,M,F,W,Y;

A123I,L,M,F,P,W,Y,V;

D124G,I,L,M,F,P,W,Y,V;

R125G,I,M,F,W,Y;

N126Q,G,I,L,M,F,P,S,T,W,Y,V;

R127A,G,I,L,M,F,P,W,Y,V;

V128I,L,M,F,P,W,Y;

I129F,W,Y;

S130A,G,I,L,M,F,P,W,Y,V;

G131A,I,L,M,F,P,W,Y,V;

E132G,I,L,M,F,W,Y;

H133M,W,V;

L134I,F,W,Y;

K136A,G,I,L,M,F,P,W,Y,V;

G145A,I,L,M,P,Y,V;

G147A,I,L,M,P,W,Y,V;

S148A,G,I,L,M,F,P,T,W,Y,V;

T149A,G,L,M,F,P,W,Y,V;

Y150W;

D152A,G,I,L,M,F,P,W,Y,V;

F153W;

K154A,G,I,L,M,F,P,W,Y,V;

H156A,G,I,L,M,F,P,W,V;

Y158W;

D164I,L,M,F,P,W,Y,V;

E167A,G,I,L,M,F,P,W,Y,V;

S168A,G,I,L,M,F,T,W,V;

R169A,G,M,P,W,Y,V;

K170A,G,I,L,M,F,P,W,Y,V;

L171W,Y;

N172A,C,Q,G,I,L,M,F,P,T,W,Y,V;

R173A,G,M,P,W,Y,V;

K176A,G,I,L,M,F,P,W,Y,V;

G179I,L,M,F,P,W,Y,V;

K180A,G,I,L,M,F,P,W,Y,V;

E185I,L,M,F,P,W,Y,V;

N188A,Q,G,L,M,F,W,V;

E189A,G,I,L,M,F,P,W,Y,V;

G191A,I,L,M,F,P,W,Y,V;

Y193W;

L196I,W;

Y198W;

D204L,M,F,P,W,Y,V;

H205A,G,I,L,M,F,P,W,Y,V;

P206 G,I,L,M,F,W,Y,V;

A209P,W,Y;

A210G,I,L,M,F,P,W,Y,V;

R214A,G,I,L,M,F,P,Y,V;

T217A,G,I,L,M,F,P,W,Y;

N221A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

E222A,G,I,L,M,F,P,W,Y,V;

K234A,G,I,M,F,P,W,Y,V;

H235A,G,I,L,M,F,P,W,Y,V;

K237A,G,I,L,M,F,W,Y,V;

S239G,I,L,M,F,P,T,Y,V;

R242G,I,L,M,F,W,Y,V;

N246A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

H247G,I,L,M,F,P,W,V;

R249A,G,I,L,M,F,P,W,Y,V;

E250A,I,L,M,P,W,Y,V;

K251G,I,L,M,F,P,W,Y,V;

T252A,G,I,L,M,F,P,W,Y,V;

G253I,L,M,F,P,W,Y;

K254A,G,I,L,M,F,P,W,Y,V;

E255A,G,I,L,M,F,W,Y,V;

F257W;

E261A,G,I,L,M,F,P,W,Y,V;

N265C,Q,I,L,M,F,P,W;

D266A,G,I,L,M,F,P,W,Y,V;

L267W,Y;

G268A,I,L,M,F,P,W,Y,V;

A269I,L,M,F,P,W,Y,V;

E271A,G,I,L,M,F,P,W,Y,V;

N272A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

N275A,C,Q,G,I,L,M,F,P,S,W,Y,V;

K276A,G,I,L,M,F,P,W,Y,V;

N278A,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

N280A,C,G,I,L,M,F,P,W,Y,V;

Y290W;

Q291A,G,I,L,M,F,P,S,T,W,Y,V;

H293A,G,I,L,M,P,W,V;

A294I,L,M,F,P,W,Y;

T297A,G,I,L,M,F,P,W,Y,V;

Q298G,I,L,M,F,P,S,T,W,Y,V;

G299A,M,P;

G300A,I,L,M,F,P,W,Y,V;

G301I,L,M,F,P,W,Y,V;

Y302W;

D303I,L,M,F,P,W,Y,V;

R305G,I,L,M,F,P,W,Y,V;

K306G,I,L,M,F,P,W,Y,V;

L308I,F,W,Y;

N309Q,G,I,L,M,F,P,S,T,W,Y,V;

G310A,I,L,M,F,P,W,Y,V;

S314A,G,I,L,M,F,P,W,Y,V;

K315A,G,I,L,M,F,P,W,Y,V;

H316A,G,I,L,M,F,P,W,V;

P317I,L,M,F,W,Y;

L318I,W,Y;

K319A,G,I,L,M,F,P,W,Y,V;

D328A,G,I,L,M,F,P,W,Y,V;

G332A,I,L,M,F,P,W,Y,V;

Q333A,C,G,I,M,F,P,S,T,Y,V;

S334G,M,F,P,W,Y;

L335I,F,W,Y;

E336A,G,I,L,M,F,P,W,Y,V;

S337A,G,I,L,M,F,P,T,W,Y,V;

T338A,G,I,L,M,F,P,W,Y,V;

Q340I,L,M,F,P,S,T,W,Y,V;

T341A,G,I,L,M,F,W,Y,V;

P345I,L,M,F,W,Y;

E355A,G,I,L,M,F,P,W,Y,V;

Y358W;

Y363W;

K370A,G,I,L,M,F,P,W,Y,V;

G371A,I,L,M,F,P,W,Y,V;

S373A,G,I,L,M,F,T,W,Y,V;

Q374A,C,G,I,L,M,F,S,T,W,Y,V;

R375A,G,I,L,M,F,P,W,V;

E376A,G,I,L,M,F,P,W,Y,V;

P378G,I,L,M,F,W,Y,V;

A379I,L,M,F,P,W,Y,V;

K381A,G,I,L,M,F,P,W,Y,V;

K389A,G,M,F,P,W,Y,V;

Q393A,C,G,I,L,M,F,P,S,T,W,Y,V;

Y394W;

Y396W;

A398I,L,M,F,W,Y,V;

Y402W;

F403W;

D404I,L,M,F,P,W,Y,V;

H405G,I,L,M,F,P,W,Y,V;

H406A,G,I,M,F,P,Y,V;

D407A,G,I,L,M,F,P,W,Y,V;

I408W,Y;

R413A,G,I,L,M,F,P,W,Y,V;

E414A,G,I,L,M,F,P,W,Y,V;

G415A,I,L,M,F,P,W,Y,V;

D416A,G,I,L,M,F,P,W,Y,V;

S417A,G,I,L,M,F,P,W,Y,V;

S418A,G,I,L,M,F,P,T,W,Y,V;

V419I,L,M,F,P,W,Y;

A420I,L,M,F,W,Y,V;

N421A,C,Q,I,L,M,F,P,S,T,W,Y,V;

S422A,G,I,L,M,F,P,T,W,Y,V;

G431A,I,L,M,F,P,W,Y,V;

P432I,L,M,F,W,Y;

G433A,I,L,M,F,P,W,Y,V;

G434A,I,L,M,F,P,W,Y,V;

A435I,L,M,F,P,W,Y,V;

K436A,G,I,L,M,F,P,W,Y,V;

R437A,G,I,L,M,F,P,W,Y,V;

Y439W;

R442A,G,I,L,M,F,P,W,Y,V;

Q443A,C,G,I,L,M,F,P,S,T,W,Y,V;

N444A,C,G,I,L,M,F,P,S,T,W,Y,V;

A445I,L,M,F,P,W,Y,V;

G446A,I,L,M,F,P,W,Y,V;

E447A,G,I,L,M,F,P,W,Y,V;

T448A,G,I,L,M,F,P,W,Y,V;

H450A,G,I,L,M,F,P,W,V;

G454A,I,L,M,F,P,W,Y,V;

N455A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

R456A,I,L,M,F,P,W,Y,V;

S457A,G,I,L,M,F,W,Y,V;

E458A,G,I,L,M,F,P,W,Y,V;

P459I,L,M,F,W,Y;

V460I,L,M,F,P,W,Y;

V461I,L,M,F,P,W,Y;

N463A,C,Q,I,L,M,F,P,S,T,W,Y,V;

S464A,G,I,L,M,F,P,T,W,Y,V;

E465A,G,I,L,M,F,P,W,Y,V;

E469A,G,I,L,M,F,P,W,Y,V;

N473Q,G,I,L,M,F,P,S,T,W,Y,V;

G474A,I,L,M,F,P,W,Y,V;

G475A,I,L,M,F,P,W,Y,V;

S476G,I,L,M,F,P,T,W,Y,V;

Q482A,C,G,I,L,M,F,S,T,W,Y,V;

R483A,G,I,L,M,F,P,W,Y,V.

General Mutations in Variants of the Invention

A variant of the invention may in one embodiment comprise one or moremodifications in addition to those outlined above. Thus, it may beadvantageous that one or more Proline (Pro) residues present in the partof the alpha-amylase variant which is modified is/are replaced with anon-Proline residue which may be any of the possible, naturallyoccurring non-Proline residues, and which preferably is an Alanine,Glycine, Serine, Threonine, Valine or Leucine.

Analogously, in one embodiment one or more Cysteine residues present inthe parent alpha-amylase may be replaced with a non-Cysteine residuesuch as Serine, Alanine, Threonine, Glycine, Valine or Leucine.

Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by anArg.

It is to be understood that the present invention encompasses variantsincorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce mutations in one ormore of the following positions (using SEQ ID NO: 8 (Termamyl) for thenumbering):

M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405, T412,in particular the following single, double or triple or multi mutations:

M15X, in particular M15T,L;

V128X, in particular V128E;

H133X, in particular H133Y;

N188X, in particular N188S,T,P;

M197X, in particular M197T,L;

A209X, in particular A209V;

M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;

M15N128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T1491;

G475R, H133Y/S187D; H133Y/A209V.

Methods for Preparing Alpha-Amylase Variants of the Invention

Several methods for introducing mutations into genes are known in theart. After a brief description of cloning of alpha-amylase-encoding DNAsequences, methods for generating mutations at specific sites within thealpha-amylase-encoding sequence will be described.

Cloning a DNA Sequence Encoding an Alpha-Amylase

The DNA sequence encoding a parent alpha-amylase may be isolated fromany cell or microorganism producing the alpha-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the alpha-amylase to be studied. Then,if the amino acid sequence of the alpha-amylase is known, homologous,labeled oligonucleotide probes may be synthesized and used to identifyalpha-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labeled oligonucleotide probecontaining sequences homologous to a known alpha-amylase gene could beused as a probe to identify alpha-amylase-encoding clones, usinghybridization and washing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase, thereby allowingclones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., The EMBO J. 3, 1984, pp. 801-805. In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al., Science 239, 1988, pp. 487-491.

Site-Directed Mutagenesis

Once an alpha-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 disclose the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNAsequences is described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Alternative methods for providing variants of the invention include geneshuffling, e.g., as described in WO 95/22625 (from Affymax TechnologiesN.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other correspondingtechniques resulting in a hybrid enzyme comprising the mutation(s),e.g., substitution(s) and/or deletion(s), in question.

Expression of Alpha-Amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding analpha-amylase variant of the invention may be any vector, which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusalpha-amylases mentioned herein comprise a preregion permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an alpha-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method ofproducing an alpha-amylase variant of the invention, which methodcomprises cultivating a host cell as described above under conditionsconducive to the production of the variant and recovering the variantfrom the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The alpha-amylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

INDUSTRIAL APPLICATIONS

The alpha-amylase variants of this invention possess valuable propertiesallowing for a variety of industrial applications. In particular, enzymevariants of the invention are applicable as a component in washing,dishwashing, and hard surface cleaning detergent compositions.

Variant of the invention with altered properties may be used for starchprocesses, in particular starch conversion, especially liquefaction ofstarch (see, e.g., U.S. Pat. No. 3,912,590, EP patent application nos.252 730 and 63 909, WO 99/19467, and WO 96/28567 all references herebyincorporated by reference). Also contemplated are compositions forstarch conversion purposes, which may beside the variant of theinvention also comprise a glucoamylase, pullulanase, and otheralpha-amylases.

Further, variants of the invention are also particularly useful in theproduction of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017hereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

Variants of the invention may also be useful for desizing of textiles,fabrics and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736,EP 119,920 hereby in corporate by reference), beer making or brewing, inpulp and paper production.

Starch Conversion

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590 and EP patent publications Nos. 252,730 and 63,909, herebyincorporated by reference.

In an embodiment the starch conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

Starch to Sugar Conversion

In the case of converting starch into a sugar the starch isdepolymerized. A such depolymerization process consists of aPre-treatment step and two or three consecutive process steps, viz. aliquefaction process, a saccharification process and dependent on thedesired end product optionally an isomerization process.

Pre-Treatment of Native Starch

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypically industrial process, the starch has to be thinned or“liquefied” so that it can be handled. This reduction in viscosity istoday mostly obtained by enzymatic degradation.

Liquefaction

During the liquefaction step, the long chained starch is degraded intobranched and linear shorter units (maltodextrins)

by an alpha-amylase. The liquefaction process is carried out at 105-110°C. for 5 to 10 minutes followed by 1-2 hours at 95° C. The pH liesbetween 5.5 and 6.2. In order to ensure optimal enzyme stability underthese conditions, 1 mM of calcium is added (40 ppm free calcium ions).After this treatment the liquefied starch will have a “dextroseequivalent” (DE) of 10-15.Saccharification

After the liquefaction process the maltodextrins are converted intodextrose by addition of a glucoamylase (e.g., AMG) and a debranchingenzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase(e.g., Promozyme™) (U.S. Pat. No. 4,560,651). Before this step the pH isreduced to a value below 4.5, maintaining the high temperature (above95° C.) to inactivate the liquefying alpha-amylase to reduce theformation of short oligosaccharide called “panose precursors” whichcannot be hydrolyzed properly by the debranching enzyme.

The temperature is lowered to 60° C., and glucoamylase and debranchingenzyme are added. The saccharification process proceeds for 24-72 hours.

Normally, when denaturing the α-amylase after the liquefaction stepabout 0.2-0.5% of the saccharification product is the branchedtrisaccharide 6²-alpha-glucosyl maltose (panose) which cannot bedegraded by a pullulanase. If active amylase from the liquefaction stepis present during saccharification (i.e., no denaturing), this level canbe as high as 1-2%, which is highly undesirable as it lowers thesaccharification yield significantly.

Isomerization

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immmobilized glucoseisomerase (such as Sweetzyme™ IT).

Ethanol Production

In general alcohol production (ethanol) from whole grain can beseparated into 4 main steps

-   -   Milling    -   Liquefaction    -   Saccharification    -   Fermentation        Milling

The grain is milled in order to open up the structure and allowing forfurther processing. Two processes are used wet or dry milling. In drymilling the whole kernel is milled and used in the remaining part of theprocess. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

Liquefaction

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by alpha-amylase.Acid hydrolysis is used on a limited basis. The raw material can bemilled whole grain or a side stream from starch processing.

Enzymatic liquefaction is typically carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and the enzyme(s) is (are) added. Then the slurry isjet-cooked at between 95-140° C., preferably 105-125° C., cooled to60-95° C. and more enzyme(s) is (are) added to obtain the finalhydrolysis. The liquefaction process is carried out at pH 4.5-6.5,typically at a pH between 5 and 6. Milled and liquefied grain is alsoknown as mash.

Saccharification

To produce low molecular sugars DP₁₋₃ that can be metabolized by yeast,the maltodextrin from the liquefaction must be further hydrolyzed. Thehydrolysis is typically done enzymatically by glucoamylases,alternatively alpha-glucosidases or acid alpha-amylases can be used. Afull saccharification step may last up to 72 hours, however, it iscommon only to do a pre-saccharification of typically 40-90 minutes andthen complete saccharification during fermentation (SSF).Saccharification is typically carried out at temperatures from 30-65°C., typically around 60° C., and at pH 4.5.

Fermentation

Yeast typically from Saccharomyces spp. is added to the mash and thefermentation is ongoing for 24-96 hours, such as typically 35-60 hours.The temperature is between 26-34° C., typically at about 32° C., and thepH is from pH 3-6, preferably around pH 4-5.

Note that the most widely used process is a simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzyme isadded together. When doing SSF it is common to introduce apre-saccharification step at a temperature above 50° C., just prior tothe fermentation.

Distillation

Following the fermentation the mash is distilled to extract the ethanol.

The ethanol obtained according to the process of the invention may beused as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutralspirits; or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid form or dried.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovering of ethanol are well known tothe skilled person.

According to the process of the invention the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alkaline alpha-amylase of the invention may also be used in theproduction of lignocellulosic materials, such as pulp, paper andcardboard, from starch reinforced waste paper and cardboard, especiallywhere re-pulping occurs at pH above 7 and where amylases facilitate thedisintegration of the waste material through degradation of thereinforcing starch. The alpha-amylase of the invention is especiallyuseful in a process for producing a papermaking pulp from starch-coatedprinted-paper. The process may be performed as described in WO 95/14807,comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylases of the invention may also be very useful in modifyingstarch where enzymatically modified starch is used in papermakingtogether with alkaline fillers such as calcium carbonate, kaolin andclays. With the alkaline alpha-amylases of the invention it becomespossible to modify the starch in the presence of the filler thusallowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

An alpha-amylase of the invention may also be very useful in textile,fabric or garment desizing. In the textile processing industry,alpha-amylases are traditionally used as auxiliaries in the desizingprocess to facilitate the removal of starch-containing size, which hasserved as a protective coating on weft yarns during weaving. Completeremoval of the size coating after weaving is important to ensure optimumresults in the subsequent processes, in which the fabric is scoured,bleached and dyed. Enzymatic starch breakdown is preferred because itdoes not involve any harmful effect on the fiber material. In order toreduce processing cost and increase mill throughput, the desizingprocessing is sometimes combined with the scouring and bleaching steps.In such cases, non-enzymatic auxiliaries such as alkali or oxidationagents are typically used to break down the starch, because traditionalalpha-amylases are not very compatible with high pH levels and bleachingagents. The non-enzymatic breakdown of the starch size does lead to somefiber damage because of the rather aggressive chemicals used.Accordingly, it would be desirable to use the alpha-amylases of theinvention as they have an improved performance in alkaline solutions.The alpha-amylases may be used alone or in combination with a cellulasewhen desizing cellulose-containing fabric or textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in WO 95/21247, U.S. Pat. No.4,643,736, EP 119,920 hereby in corporate by reference.

Commercially available products for desizing include AQUAZYME® andAQUAZYME® ULTRA from Novozymes A/S.

Beer Making

The alpha-amylases of the invention may also be very useful in abeer-making process; the alpha-amylases will typically be added duringthe mashing process.

Detergent Compositions

The alpha-amylase of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a peroxidase, another amylolytic enzyme, e.g.,another alpha-amylase, glucoamylase, maltogenic amylase, CGTase and/or acellulase, mannanase (such as MANNAWAY™ from Novozymes, Denmark)),pectinase, pectine lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likepro-teases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include ALCALASE®,SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (from NovozymesA/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECTOXP®, FN2®, FN3®, FN4® (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839. Examples of usefulalpha-amylases are the variants described in WO 94/02597, WO 94/18314,WO 96/23873, and WO 97/43424, especially the variants with substitutionsin one or more of the following positions: 15, 23, 105, 106, 124, 128,133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305,391, 408, and 444.

Commercially available alpha-amylases are DURAMYL™, LIQUEZYME™TERMAMYL™, NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A/S), RAPIDASE™ andPURASTAR™ (from Genencor International Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellu-lases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include CELLUZYME®, and CAREZYME®(Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor InternationalInc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME® (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme pre-parations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, tripho-sphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetri-aminepen-taacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the inven-tion may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the com-position may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor,in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference.

Dishwash Detergent Compositions

The enzyme of the invention mat also be used in dish wash detergentcompositions, including the following:

1) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant0.4-2.5%  Sodium metasilicate 0-20% Sodium disilicate 3-20% Sodiumtriphosphate 20-40%  Sodium carbonate 0-20% Sodium perborate  2-9%Tetraacetyl ethylene diamine (TAED)  1-4% Sodium sulphate 5-33% Enzymes0.0001-0.1%    

2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant  1-2%(e.g. alcohol ethoxylate) Sodium disilicate 2-30% Sodium carbonate10-50%  Sodium phosphonate  0-5% Trisodium citrate dihydrate 9-30%Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10%Tetraacetyl ethylene diamine (TAED)  1-2% Polyacrylate polymer 6-25%(e.g. maleic acid/acrylic acid copolymer) Enzymes 0.0001-0.1%    Perfume 0.1-0.5%  Water 5-10

3) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant0.5-2.0%  Sodium disilicate 25-40%  Sodium citrate 30-55%  Sodiumcarbonate 0-29% Sodium bicarbonate 0-20% Sodium perborate monohydrate0-15% Tetraacetyl ethylene diamine (TAED)  0-6% Maleic acid/acrylic acidcopolymer  0-5% Clay  1-3% Polyamino acids 0-20% Sodium polyacrylate 0-8% Enzymes 0.0001-0.1%    

4) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-2%Zeolite MAP 15-42%  Sodium disilicate 30-34%  Sodium citrate 0-12% Sodium carbonate 0-20%  Sodium perborate monohydrate 7-15%  Tetraacetylethylene 0-3% diamine (TAED) Polymer 0-4% Maleic acid/acrylic acidcopolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%   Sodium sulphate Balance

5) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant  1-7%Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate12-20% Monopersulphate (2 KHSO₅•KHSO₄•K₂SO₄) 15-21% Bleach stabilizer 0.1-2% Maleic acid/acrylic acid copolymer  0-6% Diethylene triaminepentaacetate,  0-2.5% pentasodium salt Enzymes 0.0001-0.1%   Sodiumsulphate, water Balance

6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANTSYSTEM Nonionic surfactant 0-1.5%  Octadecyl dimethylamine N-oxidedihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyl 0-4% dimethylamineN-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30wt. C18/C16 blend of octadecyl bis 0-5% (hydroxyethyl)amine N-oxideanhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrousC₁₃-C₁₅ alkyl ethoxysulfate with an average degree 0-10%  ofethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfate with an average degree0-5% of ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an averagedegree 0-5% of ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylatedalcohols with an 0-6.5%  average degree of ethoxylation of 9 A blend ofC₁₃-C₁₅ ethoxylated alcohols with an 0-4% average degree of ethoxylationof 30 Sodium disilicate 0-33%  Sodium tripolyphosphate 0-46%  Sodiumcitrate 0-28%  Citric acid 0-29%  Sodium carbonate 0-20%  Sodiumperborate monohydrate 0-11.5%   Tetraacetyl ethylene diamine (TAED) 0-4%Maleic acid/acrylic acid copolymer 0-7.5%  Sodium sulphate 0-12.5%  Enzymes 0.0001-0.1%   

7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION Liquid nonionicsurfactant (e.g. alcohol ethoxylates)  2.0-10.0% Alkali metal silicate 3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrier selectedfrom higher 25.0-45.0% glycols, polyglycols, polyoxides, glycolethersStabilizer (e.g. a partial ester of phosphoric acid and a  0.5-7.0%C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone)    0-1.5% Enzymes0.0001-0.1% 

8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionic surfactant(e.g. alcohol ethoxylates) 2.0-10.0% Sodium silicate 3.0-15.0% Alkalimetal carbonate 7.0-20.0% Sodium citrate  0.0-1.5% Stabilizing system(e.g. mixtures of finely divided  0.5-7.0% silicone and low molecularweight dialkyl polyglycol ethers) Low molecule weight polyacrylatepolymer 5.0-15.0% Clay gel thickener (e.g. bentonite) 0.0-10.0%Hydroxypropyl cellulose polymer  0.0-0.6% Enzymes 0.0001-0.1%  Liquidcarrier selected from higher lycols, polyglycols, Balance polyoxides andglycol ethers

9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION C₁₂-C₁₄ fattyacid 0-0.5% Block co-polymer surfactant 1.5-15.0%   Sodium citrate 0-12% Sodium tripolyphosphate  0-15% Sodium carbonate  0-8% Aluminiumtristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylatethickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%  Boric acid 0-4.0%Sodium formate 0-0.45%  Calcium formate 0-0.2% Sodium n-decydiphenyloxide disulphonate 0-4.0% Monoethanol amine (MEA) 0-1.86%  Sodiumhydroxide (50%) 1.9-9.3%  1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance

10) LIQUID AUTOMATIC DISHWASHING COMPOSITION Alcohol ethoxylate 0-20%Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20% Alkylpolyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate18-33%  Sodium citrate dihydrate 18-33%  Sodium stearate 0-2.5%  Sodiumperborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED)  0-8%Maleic acid/acrylic acid copolymer  4-8% Enzymes 0.0001-0.1%    

11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACHPARTICLES Sodium silicate 5-10%  Tetrapotassium pyrophosphate 15-25% Sodium triphosphate 0-2% Potassium carbonate 4-8% Protected bleachparticles, e.g. chlorine 5-10%  Polymeric thickener 0.7-1.5%   Potassium hydroxide 0-2% Enzymes 0.0001-0.1%    Water Balance11) Automatic dishwashing compositions as described in 1), 2), 3), 4),6) and 10), wherein perborate is replaced by percarbonate.12) Automatic dishwashing compositions as described in 1)-6) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.Uses

The present invention is also directed to methods for using analpha-amylase variant of the invention in detergents, in particularlaundry detergent compositions and dishwashing detergent compositions,hard surface cleaning compositions, and in composition for desizing oftextiles, fabrics or garments, for production of pulp and paper, beermaking, ethanol production, and starch conversion processes as describedabove.

Compositions

The invention also related to composition comprising a variant of theinvention, and in a preferred embodiment also a B. stearothermophilusalpha-amylase (BSG), in particular a variant thereof.

In another embodient the composition comprises beside a variant of theinvention a glucoamylase, in particular a glucoamylase originating fromAspergillus niger (e.g., the G1 or G2 A. niger AMG disclosed in Boel etal. (1984), “Glucoamylases G1 and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAs”, EMBO J. 3(5), p. 1097-1102, or a variant therefore, in particular a variantdisclosed in WO 00/04136 or WO 01/04273 or the Talaromyces emersonii AMGdisclosed in WO 99/28448.

In an embodiment the composition of the invention also comprises apullulanase, in particular a Bacillus pullulanase.

Materials and Methods

Materials:

Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 and alsoavailable from Novozymes A/S, Denmark.

AA560: SEQ ID NO: 12; disclosed in WO 00/60060; deposited on 25 Jan.1999 at DSMZ and assigned the DSMZ no. 12649.

LB medium (In 1 liter H₂O: 10 g bacto-tryptone, 5 g bacto-yeast extract,10 g NaCl, pH adjusted to 7.0 w. NaOH, autoclaved).

TY agar plates (In 1 liter H₂O: 16 g bacto-tryptone, 10 g bacto-yeastextract, 5 g NaCl, pH adjusted to 7.0 w. NaOH, and 15 g bacto-agar isadded prior to autoclaving).

10% Lugol solution (Iodine/Potassium iodine solution; made by 10-folddil. in H₂O of stock: Sigma Cat. no. L 6146).

Bacillus subtilis SHA273: see WO 95/10603

Detergents:

Model detergent: NP (Asia/Pacific) Model Detergent has the followingcomposition: 20% STPP (sodium tripolyphosphate), 25% Na₂SO₄, 15% Na₂CO₃,20% LAS (linear alkylbenzene sulfonate, Nansa 80S), 5% C12-C15 alcoholethoxylate (Dobanol 25-7), 5% Na₂Si2O₅, 0.3% NaCl.Omo Multi Acao (Brazil),Omo concentrated powder (EU) (Unilever)Ariel Futur liquid (EU) (Procter and Gamble)Ariel Essential (EU) (Procter and Gamble)Plasmids

pDN1528 contains the complete gene encoding Termamyl, amyL, theexpression of which is directed by its own promoter. Further, theplasmid contains the origin of replication, ori, from plasmid pUB110 andthe cat gene from plasmid pC194 conferring resistance towardschloramphenicol. pDN1528 is shown in FIG. 9 of WO 96/23874.

Methods:

Filter Screening Assays

The below assays can be used to screening of Termamyl-like alpha-amylasevariants having altered stability at high or low pH and/or under Ca²⁺depleted conditions compared to the parent enzyme and Termamyl-likealpha-amylase.

High pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith 10 micro g/ml kanamycin at 37° C. for at least 21 hours. Thecellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with glycin-NaOH buffer, pH 8.6-10.6 andincubated at room temperature (can be altered from 10-60° C.) for 15min. The cellulose acetate filters with colonies are stored on theTY-plates at room temperature until use. After incubation, residualactivity is detected on plates containing 1% agarose, 0.2% starch inglycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours at room temperature. After removal of the filters the assayplates are stained with 10% Lugol solution. Starch degrading variantsare detected as white spots on dark blue background and then identifiedon the storage plates. Positive variants are rescreened twice under thesame conditions as the first screen.

Low Calcium Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37°C. for at least 21 hours. The cellulose acetate layer is located on theTY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with carbonate/bicarbonate buffer pH 8.5-10and with different EDTA concentrations (0.001 mM-100 mM). The filtersare incubated at room temperature for 1 hour. The cellulose acetatefilters with colonies are stored on the TY-plates at room temperatureuntil use. After incubation, residual activity is detected on platescontaining 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH8.5-10. The assay plates with nitrocellulose filters are marked the sameway as the filter sandwich and incubated for 2 hours at roomtemperature. After removal of the filters the assay plates are stainedwith 10% Lugol solution. Starch degrading variants are detected as whitespots on dark blue background and then identified on the storage plates.Positive variants are rescreened twice under the same conditions as thefirst screen.

Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith 10 micro g/ml chloramphenicol at 37° C. for at least 21 hours. Thecellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter, and the nitrocellulose filter with bound variants istransferred to a container with citrate buffer, pH 4.5 and incubated at80° C. for 20 minutes (when screening for variants in the wild typebackbone) or 85° C. for 60 minutes (when screening for variants of theparent alpha-amylase). The cellulose acetate filters with colonies arestored on the TY-plates at room temperature until use. After incubation,residual activity is detected on assay plates containing 1% agarose,0.2% starch in citrate buffer, pH 6.0. The assay plates withnitrocellulose filters are marked the same way as the filter sandwichand incubated for 2 hours at 50° C. After removal of the filters theassay plates are stained with 10% Lugol solution. Starch degradingvariants are detected as white spots on dark blue background and thenidentified on the storage plates. Positive variants are re-screenedtwice under the same conditions as the first screen.

Secondary Screening

Positive transformants after rescreening are picked from the storageplate and tested in a secondary plate assay. Positive transformants aregrown for 22 hours at 37° C. in 5 ml LB+chloramphenicol. The Bacillusculture of each positive transformant and as a control a cloneexpressing the corresponding backbone are incubated in citrate buffer,pH 4.5 at 90° C. and samples are taken at 0, 10, 20, 30, 40, 60 and 80minutes. A 3 micro liter sample is spotted on an assay plate. The assayplate is stained with 10% Lugol solution. Improved variants are seen asvariants with higher residual activity (detected as halos on the assayplate) than the backbone. The improved variants are determined bynucleotide sequencing.

Stability Assay of Unpurified Variants:

Bacillus cultures expressing the variants to be analysed are grown for21 hours at 37° C. in 10 ml LB+chloramphenicol. 800 micro liter cultureis mixed with 200 micro I citrate buffer, pH 4.5. A number of 70 micro Ialiquots corresponding to the number of sample time points are made inPCR tubes and incubated at 70° C. or 90° C. for various time points(typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine. The 0 minsample is not incubated at high temperature. Activity in the sample ismeasured by transferring 20 micro I to 200 micro I of the alpha-amylasePNP-G₇ substrate MPR3 ((Boehringer Mannheim Cat. no. 1660730) asdescribed below under “Assays for Alpha-Amylase Activity”. Results areplotted as percentage activity (relative to the 0 time point) versustime, or stated as percentage residual activity after incubation for acertain period of time.

Fermentation and Purification of Alpha-Amylase Variants

A B. subtilis strain harbouring the relevant expression plasmid isstreaked on a LB-agar plate with 10 micro g/ml kanamycin from −80° C.stock, and grown overnight at 37° C. The colonies are transferred to 100ml PS-1 media supplemented with 10 micro g/ml chloamphinicol in a 500 mlshaking flask.

Composition of PS-1 medium:

Pearl sugar 100 g/l Soy Bean Meal 40 g/l Na₂HPO₄, 12 H₂O 10 g/lPluronic ™ PE 6100 0.1 g/l CaCO₃ 5 g/lThe culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions that contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

Stability Determination of Purified Variants

All stability trials of purified variants are made using the same setup. The method is as follows:

The enzyme is incubated under the relevant conditions (1-4). Samples aretaken at various time points, e.g., after 0, 5, 10, 15 and 30 minutesand diluted 25 times (same dilution for all taken samples) in assaybuffer (0.1M 50 mM Britton buffer pH 7.3) and the activity is measuredusing the Phadebas assay (Pharmacia) under standard conditions pH 7.3,37° C.

The activity measured before incubation (0 minutes) is used as reference(100%). The decline in percent is calculated as a function of theincubation time. The table shows the residual activity after, e.g., 30minutes of incubation.

Specific Activity Determination

The specific activity is determined using the Phadebas® assay(Pharmacia) as activity/mg enzyme. The manufactures instructions arefollowed (see also below under “Assay for Alpha-Amylase Activity).

Determination of Isoelectric Point

The pl is determined by isoelectric focusing (ex: Pharmacia, Ampholine,pH 3.5-9.3).

Stability Determination

The amylase stability is measured using the method as follows:

The enzyme is incubated under the relevant conditions. Samples are takenat various time points, e.g., after 0, 5, 10, 15 and 30 minutes anddiluted 25 times (same dilution for all taken samples) in assay buffer(0.1M 50 mM Britton buffer pH 7.3) and the activity is measured usingthe Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) is used as reference(100%). The decline in percent is calculated as a function of theincubation time. The table shows the residual activity after, e.g., 30minutes of incubation.

Measurement of the Calcium- and pH-Dependent Stability

Normally industrial liquefaction processes runs using pH 6.0-6.2 asliquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95° C.-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

1. lower pH than pH 6.2 and/or

2. at free calcium levels lower than 40 ppm free calcium.

Two different methods can be used to measure the alterations instability obtained by the different substitutions in the alpha-amylasein question:

Method 1.

One assay which measures the stability at reduced pH, pH 5.0, in thepresence of 5 ppm free calcium.

10 micro g of the variant are incubated under the following conditions:A 0.1 M acetate solution, pH adjusted to pH 5.0, containing 5 ppmcalcium and 5% w/w common corn starch (free of calcium). Incubation ismade in a water bath at 95° C. for 30 minutes.

Method 2.

One assay, which measure the stability in the absence of free calciumand where the pH is maintained at pH 6.0. This assay measures thedecrease in calcium sensitivity:

10 micro g of the variant were incubated under the following conditions:A 0.1 M acetate solution, pH adjusted to pH 6.0, containing 5% w/wcommon corn starch (free of calcium). Incubation was made in a waterbath at 95° C. for 30 minutes.

Oxidation Stability Determination

Raw filtered culture broths with different vatiants of the invention arediluted to an amylase activity of 100 KNU/ml (defined above) in 50 mM ofa Britton-Robinson buffer at pH 9.0 and incubated at 40° C. SubsequentlyH₂O₂ is added to a concentration of 200 mM, and the pH value isre-adjusted to 9.0. The activity is now measured after 15 seconds andafter 5, 15, and 30 minutes. The absorbance of the resulting bluesolution, measured spectrophotometrically at 620 nm, is a function ofthe alpha-amylase activity.

Washing Performance

Washing performance is evaluated by washing soiled test swatches for 15and 30 minutes at 25° C. and 40° C., respectively; at a pH in the rangefrom 9-10.5; water hardness in the range from 6 to 1

dH; Ca:Mg ratio of from 2:1 to 4:1, in different d etergent solutions(see above as described above in the Materials section) dosed from 1 to5 g/l, such as 3 g/I, dependent on the detergent with the alpha-amylasevariant in question.

The recombinant alpha-amylase variant is added to the detergentsolutions at concentrations of for instance 0.01-5 mg/l. The testswatches are soiled with orange rice starch (CS-28 swatches availablefrom CFT, Center for Test Material, Holland).

After washing, the swatches are evaluated by measuring the remission at460 nm using an Elrepho Remission Spectrophotometer. The results areexpressed as DeltaR=remission (° R□□ of the swatch washed with thealpha-amylase minus the remission of a swatch washed at the sameconditions without the alpha-amylase.

Assays for Alpha-Amylase Activity

1. Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

Alpha-amylase activity is determined by a method employing the PNP-G₇substrate. PNP-G₇ which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at 2=405 nm (400-420 nm). Kits containing PNP-G₇substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim(cat. No. 1054635).

To prepare the reagent solution 10 ml of substrate/buffer solution isadded to 50 ml enzyme/buffer solution as recommended by themanufacturer. The assay is performed by transferring 20 micro I sampleto a 96 well microtitre plate and incubating at 25° C. 200 micro Ireagent solution pre-equilibrated to 25° C. is added. The solution ismixed and pre-incubated 1 minute and absorption is measured every 30sec. over 4 minutes at OD 405 nm in an ELISA reader.

The slope of the time dependent absorption-curve is directlyproportional to the activity of the alpha-amylase in question under thegiven set of conditions.

Determination of LAS Sensitivity

The variant is incubated with different concentrations of LAS (linearalkyl benzene sulphonate; Nansa 1169/P) for 10 minutes at 40° C.

The residual activity is determined using the Phadebas® assay method orthe alternative method employing the PNP-G₇ substrate.

LAS is diluted in 0.1 M phosphate buffer pH 7.5.

The following concentrations are used:

500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm, and 10 ppm on no LAS.

The variant is diluted in the different LAS buffers to concentration of0.01-5 mg/l in a total volume of 10 ml and incubated for 10 minutes in atemperature controlled water bath. The incubation is stopped bytransferring a small aliquat into cold assay buffer. It is importantthat during activity measurement the LAS concentration is below 1 ppm,in order not to affect the activity measurement.Then the residual activity is determined in duplicate using the abovementioned Phadebas® assay or alternative method.The activity is measured after subtraction of the blank.The activity with no LAS is 100%.

EXAMPLES Example 1 Construction of Variants of the Invention andDetermination of Altered Properties

The below listed variants are constructed as described in EXAMPLE 1 ofWO 00/29560 (from Novozymes A/S) in the parent Bacillus licheniformisalpha-amylase shown in SEQ ID NO: 8.

G5A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

T6A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

G36A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

I37A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

T38A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

A39R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I42A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

A45R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

K47A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

D63A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E66A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q69A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

K70A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

G71A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

T72A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

R74A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T75A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

K76A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

T79A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

E82A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L83A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

A86R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I87A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

S89A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

R93A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T112A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

E113A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A117R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V120A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

A137R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

K213A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

G216A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

A220R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L223A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

L225A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

D226A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G227A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

R229A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D243A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V245A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

F279A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

S282A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

T311A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

V321A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

V324A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

L352A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

T353A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

R354A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G357A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

V361A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

F362A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

G364A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

G368A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

A390R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A395R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G397A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

Q399A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H400A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

D401A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A425R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D451A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I452A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

T453A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

G466A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

G468A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

F470A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

H471A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

S478A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V

L7A,R,N,D,C,Q,E,G,H,K,M,P,S,Y,V;

M8C;

Q9A,R,N,D,C,G,H,M,P,S,T,W,Y,V;

F11A,N,D,C,Q,G,H,I,L,M,P,S,T,W,Y,V;

E12A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G19A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

H21A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

W22A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

L25A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

L32A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

V40A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

W41A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

Y46A,R,D,C,G,K,M,P,W;

G48R,N,D,C,Q,E,H,K,M,F,P,W,Y;

G55A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

G57R,N,D,C,Q,E,H,K,M,P,W;

A58R,N,D,C,Q,E,G,H,K,M,S,T,W,Y;

D60A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y77A,R,D,C,G,K,M,P,W;

I95A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

V97A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

Y98A,R,D,C,G,K,M,P,W;

G99R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

D100A,R,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

V101A,N,C,Q,G,I,L,M,P,S,T,W,Y;

V102N,D,C,Q,E,H,I,L,M,F,P,W,Y;

I103A,N,D,C,Q,E,G,M,P,S,W,Y;

H105A,N,C,Q,G,I,L,M,P,S,T,Y,V;

G107R,N,D,Q,E,H,K,M,F,P,W,Y;

V115R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

V118R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

I135A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

T139A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

F141A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

F143A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

S151A,R,N,D,C,Q,E,G,H,K,M,P,T,Y,V;

H159A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

F160A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

D161A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G162A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

T163A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

D166A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y175A,R,D,C,G,K,M,P,W;

F177A,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

D183A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V186A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

S187A,R,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

N192A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A199R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D200A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D202A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y203A,R,D,C,G,K,M,P;

V208A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y;

I212A,N,C,Q,G,H,M,P,S,T,V;

W215A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

Y219A,R,D,C,G,K,M,P,W;

F228A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

L230A,R,N,D,C,Q,E,G,M,P,S,T,W,Y,V;

V233R,N,C,Q,E,G,H,I,K,M,P,S,T,W,Y;

I236A,C,Q,G,H,M,P,S,T,V;

F238A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

F240A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

L241A,N,C,Q,G,H,P,S,T,V;

W244A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

V248A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

M256C;

T258A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

V259A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y;

A260R,N,D,C,Q,E,H,I,L,K,M,F,P,T,W,Y,V;

Y262A,R,D,C,G,K,M,P,W;

L270A,N,C,Q,G,I,M,F,P,S,T,W,Y,V;

Y273A,R,D,C,G,K,M,P;

L274A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

T277A,R,N,D,C,Q,E,G,H,K,M,P,S,W,Y,V;

H281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

V283A,R,N,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y;

F284A,R,N,D,C,Q,E,G,I,L,K,M,P,S,T,Y,V;

D285A,R,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

V286A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y;

P287R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

L288A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

H289A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

F292A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

A295R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S296A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

M304C;

L307A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

V312A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y;

V313A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

S320R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

T322R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

F323A,R,N,D,C,Q,E,G,I,L,K,M,P,S,T,W,Y,V;

D325A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N326A,R,C,Q,E,G,M,P,S,T,W;

H327A,R,C,G,I,L,K,M,P,S,T,W,Y,V;

T329A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

P331N,D,C,Q,E,G,H,I,L,M,F,S,T,W,Y,V;

V339A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

F343A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

K344A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

L346A,R,N,D,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

A347R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

A349R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

F350A,R,N,C,Q,G,H,I,L,K,M,P,S,T,Y,V;

P359R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

Q360N,D,G,H,I,L,M,F,P,S,T,W,Y,V;

T369A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

I377A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

L380A,R,N,D,C,Q,E,G,H,K,P,S,W,Y,V;

I387A,R,N,D,C,Q,E,G,H,L,K,M,P,S,T,W,Y,V;

V409N,C,Q,E,G,H,M,P,S,T,W,Y;

G410A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

W411A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

T412R,N,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

G423A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

L424A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

A426R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L427A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

I428A,N,D,Q,E,G,H,M,F,P,S,W,Y,V;

T429A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

D430A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

M438C;

V440R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

G441A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

W449A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

I462A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

V472A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

V477A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

I479A,R,N,D,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

Y480A,R,D,C,G,K,M,P,W;

V481A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y.

A1R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

N2R,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

L3A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y;

N4A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

W13R,N,D,C,Q,E,G,H,K,M,P,S,T;

Y14A,R,D,C,G,K,M,P,W;

P16R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

N17A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D18A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q20A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R23A,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

R24A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q26A,D,C,E,G,H,I,L,M,F,P,S,T,W,V;

E34A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

H35A,R,N,D,C,Q,E,G,K,M,F,P,S,T,W,Y,V;

T49A,C,G,H,P;

S50A,R,N,C,Q,E,G,H,K,M,F,P,W;

Q51A,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

A52R,D,C,Q,E,G,H,K,P;

D53A,C,G,H,K,M,P;

L61A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,Y;

Y62A,R,D,C,G,K,M,P;

F67A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

H68A,R,D,C,E,G,I,L,K,M,F,P,S,T,W,Y,V;

V73A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y;

Q84A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S85A,R,N,C,E,G,H,I,L,K,M,F,P,T,W,Y,V;

K88A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

H91A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

S92A,R,N,D,C,Q,E,G,H,I,L,M,F,P,T,W,Y,V;

N96A,R,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

K106A,N,D,C,Q,E,G,H,I,L,M,P,S,T,Y,V;

G108R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

D114A,N,C,Q,E,G,H,K,F,P,S,T,W,Y;

T116A,R,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

E119A,R,N,D,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D121A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

P122R,N,Q,G,H,I,L,M,F,S,T,W,Y,V;

A123N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

D124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

R125N,Q,E,G,I,K,M,F,S,T,W,Y;

N126Q,G,H,I,L,M,F,P,S,T,W,Y,V;

R127A,N,D,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V128A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,W,Y;

I129A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

S130A,R,N,D,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

G131A,R,N,D,C,Q,H,I,L,K,M,F,P,S,T,W,Y,V;

E132R,N,D,C,Q,G,H,I,L,K,M,F,S,W,Y;

H133R,N,D,C,M,T,W,V;

L134A,N,D,C,E,G,H,I,K,M,F,P,S,T,W,Y,V;

K136A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

W138R,N,D,Q,E,G,I,K,M,P,S,T,V;

G145A,R,N,D,C,Q,E,H,I,L,K,M,P,S,T,Y,V;

G147A,R,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

S148A,R,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

T149A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

Y150A,D,C,G,M,P,W;

D152A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

F153A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

K154A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

W155A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

H156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

W157R,I,L,M,F,P,S,T,Y,V;

Y158R,M,P,W;

D164R,I,L,M,F,P,S,T,W,Y,V;

W165A,N,D,C,Q,E, H,I,L,K,M,F,P,S,T,Y,V;

E167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S168A,R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,V;

R169A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

K170A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

L171A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

N172A,D,C,Q,E,G,I,L,M,F,P,T,W,Y,V;

R173A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

K176A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

G179D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

K180A,G,I,L,M,F,P,W,Y,V;

A181G;

W182A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

W184I,L,M,F,P,W,Y,V;

E185R,I,L,M,F,P,S,T,W,Y,V;

N188A,R,Q,G,H,L,M,F,W,V;

E189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

G191A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

Y193A,R,G,M,P,W;

L196A,N,Q,G,H,I,M,P,S,T,W,V;

Y198A,R,G,M,P,W;

D204R,L,M,F,P,T,W,Y,V;

H205A,G,I,L,M,F,P,W,Y,V;

P206R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

A209R,P,S,W,Y;

A210R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

R214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

T217A,R,N,D,C,Q,G,H,I,L,M,F,P,S,W,Y;

W218A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

N221A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E222A,R,N,D,C,G,H,I,L,K,M,F,P,S,W,Y,V;

K234A,D,C,G,H,I,M,F,P,S,T,W,Y,V;

H235A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

K237A,G,H,I,L,M,F,W,Y,V;

S239G,H,I,L,M,F,P,T,Y,V;

R242G,I,L,M,F,S,T,W,Y,V;

N246A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H247R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

R249A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E250A,R,N,D,C,H,I,L,K,M,P,T,W,Y,V;

K251R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

T252A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

G253R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

K254A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

E255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

F257A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

E261A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

W263A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,Y,V;

N265C,Q,E,H,I,L,M,F,P,W;

D266A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L267A,R,N,D,C,Q,E,G,H,K,P,S,T,W,Y,V;

G268A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

A269N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E271A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N272A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N275A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

K276A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

N278A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N280A,R,D,C,E,G,H,I,L,K,M,F,P,W,Y,V;

Y290W;

Q291A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

H293A,R,N,G,I,L,M,P,S,T,W,V;

A294R,N,Q,G,H,I,L,M,F,P,S,T,W,Y;

T297A,G,H,I,L,M,F,P,W,Y,V;

Q298G,H,I,L,M,F,P,S,T,W,Y,V;

G299A,H,M,P,S,T;

G300A,C,H,I,L,M,F,P,T,W,Y,V;

G301N,Q,H,I,L,M,F,P,S,T,W,Y,V;

Y302R,M,P,W;

D303R,I,L,M,F,P,S,T,W,Y,V;

R305G,I,L,M,F,P,S,T,W,Y,V;

K306Q,G,H,I,L,M,F,P,S,T,W,Y,V;

L308A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

N309Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G310A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

S314A,D,C,E,G,I,L,M,F,P,W,Y,V;

K315A,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

H316A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

P317R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

L318A,R,N,D,C,Q,E,G,H,I,K,P,S,W,Y,V;

K319A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

D328A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G332A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

Q333A,N,D,C,G,I,M,F,P,S,T,Y,V;

S334R,N,C,Q,E,G,H,K,M,F,P,W,Y;

L335R,D,C,Q,E,H,I,K,M,F,P,W,Y,V;

E336A,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

S337A,R,N,C,Q,E,G,H,I,L,M,F,P,T,W,Y,V;

T338A,R,N,C,Q,G,H,I,L,K,M,F,P,S,W,Y,V;

Q340I,L,M,F,P,S,T,W,Y,V;

T341A,R,D,C,G,H,I,L,K,M,F,W,Y,V;

W342R,I,L,M,F,P,S,T,Y,V;

P345R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,Y,V;

E355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y358A,R,D,C,G,K,M,P,W;

Y363A,R,D,C,G,K,M,P,W;

K370A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

G371A,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

S373A,R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,Y,V;

Q374A,N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

R375A,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,V;

E376A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

P378R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

A379R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

K381A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

K389A,D,C,G,H,M,F,P,S,T,W,Y,V;

Q393A,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y394A,R,D,C,G,K,M,P,W;

Y396A,R,D,C,G,K,M,P,W;

A398R,N,D,C,Q,E,G,H,I,L,M,F,W,Y,V;

Y402R,C,G,K,M,P,W;

F403A,R,N,Q,G,H,I,L,M,P,S,T,W,Y,V;

D404R,I,L,M,F,P,S,T,W,Y,V;

H405R,G,I,L,M,F,P,W,Y,V;

H406A,R,G,I,M,F,P,Y,V;

D407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

I408A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

R413A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E414A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G415A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

D416A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

S417A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

S418A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

V419A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y;

A420N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

N421A,R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

S422A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

G431A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

P432G,H,I,L,M,F,S,T,W,Y,V;

G433A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

G434A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

A435Q,G,H,I,L,M,F,P,T,W,Y,V;

K436A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

R437A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Y439A,R,D,C,G,K,M,P,W;

R442A,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

Q443A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

N444A,C,G,H,I,L,M,F,P,S,T,W,Y,V;

A445R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G446A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

E447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

T448A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

H450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

G454A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

N455A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R456A,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

S457A,R,N,D,C,Q,E,G,H,I,L,K,M,F,W,Y,V;

E458A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

P459R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y,V;

V460A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

V461A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y;

N463A,R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

S464A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

E465A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

W467A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

E469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N473Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G474A,R,H,I,L,M,F,P,W,Y,V;

G475A,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

S476G,H,I,L,M,F,P,T,W,Y,V;

Q482A,N,D,C,G,H,I,L,M,F,S,T,W,Y,V;

R483A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V.

The variants are tested for altered substrate specificity, substratebinding, substrate cleavage pattern, thermal stability, pH activityprofile, pH stability profile, stability towards oxidation, Ca²⁺dependency, reduced LAS sensitivity, reduced and increased pl andimproved wash performance, and specific activity as described in the“Materials & Methods” section above.

Example 2 Construction of Variants of the Invention and Determination ofAltered Properties

The below listed variants are constructed as described in EXAMPLE 1 ofWO 00/37626 (from Novozymes A/S) in the parent Bacillus licheniformisalpha-amylase shown in SEQ ID NO: 8. The alterations of the variantsare, as specified in the list below, insertion of an amino aciddownstream of the amino acid which occupies the position, or deletion ofthe amino acid which occupies the position.

A1 insertion;

L3 insertion;

N4 insertion;

N17 insertion;

D18 insertion;

Q20 insertion;

R23 insertion;

R24 insertion;

D28 insertion;

Y56 insertion;

L61 insertion or deletion;

Y62 insertion;

F67 insertion or deletion;

H68 insertion;

K80 insertion or deletion;

G81 insertion or deletion;

Q84 insertion;

S85 insertion;

H91 insertion or deletion;

S92 insertion or deletion;

K106 insertion or deletion;

D110 insertion or deletion;

D114 deletion;

E119 insertion or deletion;

D121 insertion;

P122 insertion;

A123 insertion;

D124 insertion;

R125 insertion;

N126 insertion;

R127 insertion;

I129 insertion;

G131 insertion;

L134 insertion;

K136 insertion;

N172 insertion;

E185 insertion;

L196 insertion or deletion;

P206 insertion or deletion;

T217 insertion;

W218 insertion;

D231 insertion or deletion;

A232 insertion or deletion;

H235 insertion or deletion;

N246 insertion;

H247 insertion;

R249 insertion;

K251 insertion;

F257 insertion or deletion;

N278 insertion;

G310 insertion or deletion;

H316 insertion;

P317 insertion;

D328 insertion or deletion;

G332 insertion or deletion;

E355 insertion or deletion;

Y358 insertion;

Y363 insertion;

Y367 insertion;

K370 insertion;

S373 insertion;

R375 insertion;

E376 insertion;

K381 insertion;

H382 insertion;

R391 insertion or deletion;

Y396 insertion;

R413 insertion or deletion;

E414 insertion or deletion;

G415 insertion or deletion;

D416 insertion;

S417 insertion;

S418 insertion;

V419 insertion;

A420 insertion;

N421 insertion;

S422 insertion or deletion;

Y439 insertion;

A445 insertion or deletion;

G446 insertion or deletion;

T448 insertion or deletion;

H450 insertion;

G454 insertion or deletion;

N455 insertion;

E458 insertion;

P459 insertion;

V460 insertion;

V461 insertion;

N463 insertion;

S464 insertion;

E465 insertion;

W467 insertion;

L7 insertion or deletion;

M8 insertion;

Y10 insertion;

F11 insertion;

E12 insertion or deletion;

M15 insertion;

G19 insertion;

H21 insertion;

W22 insertion;

L25 insertion;

V40 insertion or deletion;

W41 insertion;

P43 insertion or deletion;

P44 insertion or deletion;

Y46 insertion;

G55 insertion;

Y59 insertion;

Y77 insertion;

G78 insertion or deletion;

L90 insertion or deletion;

I95 insertion;

V97 insertion;

Y98 insertion;

G99 insertion;

D100 insertion;

V101 insertion;

V102 insertion;

H105 insertion or deletion;

A109 insertion or deletion;

V115 insertion or deletion;

V118 insertion or deletion;

I135 insertion;

T139 insertion or deletion;

F141 insertion or deletion;

Y195 insertion;

V208 insertion or deletion;

W215 insertion;

Y219 insertion;

I236 insertion or deletion;

F238 insertion or deletion;

F240 insertion or deletion;

W244 insertion;

V248 insertion;

M256 insertion;

T258 insertion or deletion;

V259 insertion or deletion;

V312 insertion or deletion;

V313 insertion or deletion;

S320 insertion;

T322 insertion or deletion;

F323 insertion or deletion;

D325 insertion or deletion;

N326 insertion;

H327 insertion or deletion;

Q330 insertion or deletion;

P331 insertion or deletion;

Y348 insertion;

A349 insertion or deletion;

F350 insertion or deletion;

P359 insertion or deletion;

Q360 insertion;

D365 insertion or deletion;

M366 insertion;

T369 insertion;

I377 insertion;

I384 insertion or deletion;

L388 insertion or deletion;

G423 insertion or deletion;

L424 insertion or deletion;

M438 insertion;

G441 insertion or deletion;

W449 insertion;

I462 insertion;

I479 insertion or deletion;

Y480 insertion;

V481 insertion or deletion.

The variants are tested for altered substrate specificity, substratebinding, substrate cleavage pattern, thermal stability, pH activityprofile, pH stability profile, stability towards oxidation, Ca²⁺dependency, reduced and increased pl and improved wash performance,specific activity as described in the “Materials & Methods” sectionabove.

The invention claimed is:
 1. An isolated variant polypeptide havingalpha-amylase activity, the variant polypeptide comprising: an aminoacid sequence having at least 95% amino acid sequence identity to SEQ IDNO: 8; and (ii) a substitution that introduces an isoleucine residue ata position corresponding to position 185 of SEQ ID NO:
 8. 2. The variantpolypeptide of claim 1, wherein the variant polypeptide comprises anamino acid sequence having at least 96% sequence identity to SEQ ID NO:8.
 3. The variant polypeptide of claim 1, wherein the variantpolypeptide comprises an amino acid sequence having at least 97%sequence identity to SEQ ID NO:
 8. 4. The variant polypeptide of claim1, wherein the variant polypeptide comprises an amino acid sequencehaving at least 98% sequence identity to SEQ ID NO:
 8. 5. The variantpolypeptide of claim 1, wherein the variant polypeptide comprises theamino acid sequence of SEQ ID NO: 8 having a substitution thatintroduces a prolinc an isoleucine residue at a position correspondingto position 185 of SEQ ID NO:
 8. 6. The variant polypeptide of claim 1,wherein the variant polypeptide consists of the amino acid sequence ofSEQ ID NO: 8 having an E185P a substitution that introduces anisoleucine residue at a position corresponding to position 185 of SEQ IDNO:
 8. 7. A detergent composition comprising a surfactant, and thevariant polypeptide of claim
 1. 8. A detergent composition comprisingthe variant polypeptide of claim
 1. 9. The detergent composition ofclaim 8, wherein the variant polypeptide consists of the amino acidsequence of SEQ ID NO: 8 having a substitution that introduces anisoleucine residue at a position corresponding to position 185 of SEQ IDNO:
 8. 10. A composition for starch conversion comprising the variantpolypeptide of claim
 1. 11. The composition for starch conversion ofclaim 10, wherein the variant polypeptide comprises an amino acidsequence having at least 97% sequence identity to SEQ ID NO:
 8. 12. Thecomposition for starch conversion of claim 10, wherein the variantpolypeptide comprises an amino acid sequence having at least 99%sequence identity to SEQ ID NO:
 8. 13. The composition for starchconversion of claim 10, wherein the variant polypeptide consists of theamino acid sequence of SEQ ID NO: 8 having a substitution thatintroduces an isoleucine residue at a position corresponding to position185 of SEQ ID NO:
 8. 14. A process for producing a fermentation product,comprising: (a) saccharifying a cellulosic material with an enzymecomposition in the presence of the variant polypeptide of claim 1; (b)fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.
 15. Theprocess of claim 14, wherein the fermentation product is ethanol. 16.The process of claim 14, wherein the variant polypeptide comprises anamino acid sequence having at least 99% amino acid sequence identity toSEQ ID NO:
 8. 17. The process of claim 14, wherein the variantpolypeptide consists of the amino acid sequence of SEQ ID NO: 8 having asubstitution that introduces an isoleucine residue at a positioncorresponding to position 185 of SEQ ID NO:
 8. 18. An alpha-amylasevariant comprising a substitution at a position corresponding toposition 185 of the polypeptide of SEQ ID NO: 8, wherein thesubstitution is E185I and the variant has alpha-amylase activity,wherein the variant comprises an amino acid sequence having at least 99%sequence identity to SEQ ID NO: 8.